Heavy Water and Hydrostatic Pressure Effects on Tetrahymena pyriformis1

SYNOPSIS. Heat-synchronized cultures of Tetrahymena pyriformis strain GL subjected to pulses of high hydrostatic pressure (10,000 psi for 2 min) had increasing division delays during the 1st 40 min after the last heat shock (40 min after heat treatment). Pressure treatment during the subsequent 10-min interval disrupted cell synchrony. Comparable pressures applied to the cells at later stages, before the 1st synchronous division, caused negligible division delay. Continuous exposure to 10% (v/v) heavy water hardly affected division; higher concentrations delayed or blocked division. Ten-min pulses with heavy water (40%, 50%, 70%) resulted in increasing division delays depending on the stage of the cell cycle during which the heavy water was applied. Amelioration of the division-delaying effects of pressure was observed in cells treated simultaneously with pressure (3,000 psi for 30 min), and 30% D₂O. The results are consistent with the hypothesis that some of the pressure and D₂O effects could be attributed to changes in the sol-gel state of the cytoplasm.     

The effect of deuterium oxide on light-sensitive Paulownia tomentosa seed germination

The effect of deuterium oxide on the germination of light-sensitive Empress tree (Paulownia tomentosa) seeds was studied. Kinetics of germination in both light- and gibberellic acid (GA₃)-induced seeds was decelerated by increasing concentrations of deuterium oxide (D₂O) in the medium. Deuterium oxide in increasing concentrations also changed the duration of the light necessary for germination, from 5 min in H₂O to more than 3 days in 60% D₂O. A far-red (FR) pulse reversed the effect of inductive, prolonged red (R) irradiation; maximum germination could subsequently be restored by only 5 min of red light. The escape from FR reversibility was delayed from 1 day in water to 2 or 3.5 days in D₂O. The light reactions of D₂O-treated seeds resemble those of skotodormant seeds of the same species.     

The thermal stability of turnip yellow mosaic virus under hydrostatic pressure

The thermal stability of an isometric plant virus, Turnip Yellow Mosaic Virus (TYMV), has been investigated at low and high hydrostatic pressure, using small angle neutron scattering. Contrast variation allowed us to separately observe the structural changes of the protein capsid and the RNA core. The experiments were performed in 0.05M Tris buffer at pD = 8.0 and in 0.05M bis-Tris buffer at pD = 6.0 containing different H₂O/D₂O mixtures (40% and 70% D₂O). It was found that hydrostatic pressure enhances the stability of TYMV. The thermally induced uncoating of RNA as well as structural transitions of the protein capsid are shifted to higher temperature upon increasing the pressure from 5 × 10⁶ Pa to 2 × 10⁸ Pa.     

Oscillatory Transpiration and Water Uptake of Avena Plants

The action of D₂O on oscillatory transpiration of Avena plants was investigated. D₂O affects the amplitude and the period of the oscillations when given as a root medium to intact plants. The period is then dependent on the amplitude. From such experiments it is not possible to conclude whether the period change is simply due to the changed amplitude or to a change in the stomatal parameters. When given to xylem compressed, excised plants without roots, the D₂O hardly affects the amplitude of the oscillations but the period is increased. Thus, the period of the self-sustained transpiratory oscillations is lengthened by D₂O action on the stomatal parameters. Phase and amplitude changes of the oscillatory transpiration caused by short D₂O pulses given both to intact and excised plants, are discussed. The following conclusion is emphasized: a substance which affects the root system can also cause profound changes in the stomatal water regulation.     

Poa annua shows inter-generational differences in response to elevated CO2

Inter-generational effects on the growth of Poa annua (L.) in ambient and elevated atmospheric CO₂ conditions (350 and 550 μl l^–1, respectively) were studied in two different experiments. Both experiments showed similar results. In a greenhouse experiment growth, measured as the numbers of tillers produced per week, was compared for plants grown from first and second generation seeds. Second generation seeds were obtained from plants grown for one whole generation in either ambient or elevated atmospheric CO₂ (‘ambient’ and ‘elevated’ seeds, respectively). First generation plants and second generation ‘ambient’ plants did not respond to elevated CO₂. Second generation ‘elevated’ plants produced significantly more tillers in elevated CO₂. In the second experiment model terrestrial ecosystems growing in the Ecotron and which included Poa annua were used. Above-ground biomass after one and two generations of growth were compared. At the end of Generation 1 no difference was found in biomass production while at the end of Generation 2 biomass increased in elevated CO₂ by 50%. The implications for climate change research are discussed.     

Properties ofEscherichia coli grown in deuterated media

The effects on a number of parameters of transferringEscherichia coli between protonated and deuterated media were studied; these included growth, oxygen consumption and the synthesis of DNA, RNA, total protein, and β-galactosidase. Similar measurements were made on cells fully adapted to growth on deuterated media. The amino acid compositions of deuterated and protonated cellular protein were similar, but in deuterated cells the ratio protein: DNA was doubled. Deutero- and protio-β-galactosidase had similarK _M values and turnover numbers in D₂O and H₂O. The kinetics of β-galactosidase synthesis were not changed by deuteration, but it was found that lower concentrations of inducer were required to achieve particular levels of induction. Brief exposure to inducer in one medium, followed by removal of inducer and expression of enzyme-forming-potential in either D₂O or H₂O, showed that mRNA synthesized by deuterated cells was translated equally well in both media. mRNA synthesized by protonated cells was translated about twice as efficiently in H₂O. Inducible strains (but not a regulator constitutive) lost the capacity to synthesize enzymically active β-galactosidase after more than 100 generations in D₂O-acetate. The defect persisted when such cells were grown in H₂O-acetate, but enzyme activity was restored by growth in H₂O-glycerol. The failure to produce active enzyme was not due to a failure of the induction mechanism; gel electrophoresis revealed the presence of an inactive protein species. The nature of adaptation to deuteration is discussed.     

Production of high levels of poly‐3‐hydroxybutyrate in plastids of Camelina sativa seeds

Poly‐3‐hydroxybutyrate (PHB) production in plastids of Camelina sativa seeds was investigated by comparing levels of polymer produced upon transformation of plants with five different binary vectors containing combinations of five seed‐specific promoters for expression of transgenes. Genes encoding PHB biosynthetic enzymes were modified at the N‐terminus to encode a plastid targeting signal. PHB levels of up to 15% of the mature seed weight were measured in single sacrificed T₁ seeds with a genetic construct containing the oleosin and glycinin promoters. A more detailed analysis of the PHB production potential of two of the best performing binary vectors in a Camelina line bred for larger seed size yielded lines containing up to 15% polymer in mature T₂ seeds. Transmission electron microscopy showed the presence of distinct granules of PHB in the seeds. PHB production had varying effects on germination, emergence and survival of seedlings. Once true leaves formed, plants grew normally and were able to set seeds. PHB synthesis lowered the total oil but not the protein content of engineered seeds. A change in the oil fatty acid profile was also observed. High molecular weight polymer was produced with weight‐averaged molecular weights varying between 600 000 and 1 500 000, depending on the line. Select lines were advanced to later generations yielding a line with 13.7% PHB in T₄ seeds. The levels of polymer produced in this study are the highest reported to date in a seed and are an important step forward for commercializing an oilseed‐based platform for PHB production.     

Effect of maturation duration on desiccation tolerance in hybrid larch (Larix×leptoeuropaea dengler) somatic embryos

Summary Cotyledonary somatic embryos ofLarix × leptoeuropaea that developed after various maturation times on media containing abscisic acid showed different frequencies of conversion into plants. Drying of these somatic embryos under high relative humidity (RH) before germination improved plantlet recovery and eliminated differences in the performance of somatic embryos matured for different times. However, dehydration of somatic embryos under 98% RH to a water content below that of zygotic embryos excised from mature seeds (0.97 and 1.36 g H₂O/g dry weight, respectively) showed a strong positive correlation between longer maturation time and desiccation tolerance. Drying somatic embryos at 4° C under 59% RH for 1 wk resulted in desiccation to a water content of 0.30 g H₂O/g dry weight, which was the closest to the hydration state of zygotic embryos in dried, stored seeds (0.20 g H₂O/g dry weight). Under this condition, only somatic embryos matured for 5 wk germinated and produced plantlets at a relatively high frequency (73 and 41%, respectively).     

Very long chain and hydroxylated fatty acids in offspring of somatic hybrids between Brassica napus and Lesquerella fendleri

 Offspring of somatic hybrids between the zero-erucic acid rapeseed cv Hanna and Lesquerella fendleri were analysed regarding their fatty acid profiles. In the first back-cross generation one plant was found that produced a seed containing up to 16.5% erucic acid and 15% eicosaenoic acid (Line 1), as well as a seed having 4.3% ricinoleic acid (Line 2). This was interpreted as due to a contribution of elongase and hydroxylase genes from the L. fendleri genome since these two fatty acids are not produced in the recipient rapeseed cultivar Hanna. Crosses between Line 1 and cv Hanna resulted in the production of seeds with 35% erucic acid (F₂). Furthermore, crosses between the F₂ plants and the rapeseed cultivar Gulle, producing 35% erucic acid in the seeds, resulted in F₃ seeds with 48% erucic acid. The highest amount of erucic acid, 61.5%, was found in the F₆ generation after crossing Line 1 with a high erucic acid rapeseed line, HEAR, followed by self-fertilisation for two generations. When performing Southern-blot analysis on the F₆ plants, seven of the nine analysed plants hybridised with the L. fendleri species-specific repetitive probe. The presence of the hydroxylase gene was also observed in the F₆ generation of Line 1 according to Southern-blot analysis. Hybridisation with a hydroxylase probe was seen although no hydroxy fatty acids could be detected in any of the F₆ plants. In parallel, Line 2 was crossed with HEAR cv Gulle and self fertilised. No hydroxy fatty acids were detected in the F₂ generation of Line 2 and no specific hybridisation patterns could be found in the Southern-blot analysis. Received: 12 December 1998 / Accepted: 4 January 1999     

Epigenetic Variation Induced in Triticum aestivum L. by Nicotinic Acid

The effect of nicotinic acid (NA) on hereditary traits of spring common wheat cultivar Kazakhstanskaya 126 (K.126) were studied under the laboratory and field conditions. Treatment of seeds and vegetating plants with 0.01–0.1% NA (aqueous solution) induced heritable epigenetic changes in wheat. As a result, strong tall plants with the long productive spike, large seeds, and several quantitative and qualitative characters other than in the original cultivar were obtained in the second and further generations after treatment. Crosses of changed plants with each other did not result in segregation with respect to leaf downiness or anthocyan stem color in F₂–F₄, suggesting the same epigenetic state of genes responsible for changed characters. In crosses with the original cultivar, characters of the changed plants always dominated in F₁. Basing on the current views, the changes were attributed to a transition of thehl ₁ and pc recessive marker genes into new, dominant epiallelic states Hl ₁ and Pc, which respectively determine downy leaves and the colored stem. The NA effect was specific, since only one type of the variation was observed. The changed characters were stable, and no reversion to the original phenotype was detected in 57 generations.     

Seed germination and in vitro propagation of Maytenus canariensis through regeneration of adventitious shoots from axillary and apical buds

Seed germination and micropropagation protocols of the medicinal species Maytenus canariensis (Loes.) G. Kunkel & Sunding were optimized. In vitro seed germination occurred (86 to 94.7 %) only after treatment of the seeds with H₂SO₄, followed by surface sterilization and culture on solid nutrient medium without any growth regulators. Micropropagation failed when explants were taken from mature trees, and browning of the nutrient medium frequently occurred despite testing many growth media. Nonetheless, adventitious shoot regeneration was achieved employing axillary or apical buds taken from 2–2.5 months old plantlets obtained after in vitro germination of seeds, following culture on nutrient media supplemented with benzylaminopurine, kinetin and naphthalenacetic acid (NAA), attaining up to 3.9 shoots per explant, after 4–6 months. Root induction was best on a medium containing 4.0 mg dm⁻³ NAA, achieving a 100 % induction. After hardening of rooted plants, survival after transfer to soil was 71.43 %.     

Physiological Studies with Heavy Water

The absorption of H₂O and D₂O was studied in seeds with different reserve food materials represented by barley and rice (starch), linseed and mustard (fat) and mung, and kidney beans (protein). Respiration rate and respiratory quotient during absorption were also measured. The results indicate that the water absorption profiles of the starchy and fatty seeds are essentially similar but differ from that of The proteinaceous type. Initial hydration is relatively incomplete in D₂O in all The seeds to varying degrees the latter being maximum in The proteinaceous and minimum in The starchy seeds. The respiration rate is lower in D₂O while The R.Q. values are higher for The first hour but lower later on. The results, in part, help explain The varying effects of D₂O on The germination of different types of seeds. These studies also support The earlier reported preference (Cope et al. 1965) by the higher plant systems for protium.     

Reduction of Escherichia coli O157:H7 on radish seeds by sequential application of aqueous chlorine dioxide and dry‐heat treatment

Aims: To assess the effectiveness of sequential treatments of radish seeds with aqueous chlorine dioxide (ClO₂) and dry heat in reducing the number of Escherichia coli O157:H7. Methods and Results: Radish seeds containing E. coli O157:H7 at 5·5 log CFU g^?1 were treated with 500 μg ml^?1 ClO₂ for 5 min and subsequently heated at 60°C and 23% relative humidity for up to 48 h. Escherichia coli O157:H7 decreased by more than 4·8 log CFU g^?1 after 12 h dry‐heat treatment. The pathogen was inactivated after 48 h dry‐heat treatment, but the germination rate of treated seeds was substantially reduced from 91·2 ± 5·0% to 68·7 ± 12·3%. Conclusions: Escherichia coli O157:H7 on radish seeds can be effectively reduced by sequential treatments with ClO₂ and dry heat. To eliminate E. coli O157:H7 on radish seeds without decreasing the germination rate, partial drying of seeds at ambient temperature before dry‐heat treatment should be investigated, and conditions for drying and dry‐heat treatment should be optimized. Significance and Impact of the study: This study showed that sequential treatment with ClO₂ and dry‐heat was effective in inactivating large numbers of E. coli O157:H7 on radish seeds. These findings will be useful when developing sanitizing strategies for seeds without compromising germination rates.     

Genetic transformation of NERICA,interspecific hybrid rice between <Emphasis Type="Italic">Oryza glaberrima</Emphasis> and <Emphasis Type="Italic">O. sativa</Emphasis>, mediated by <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>

We developed an efficient gene transfer method mediated by Agrobacterium tumefaciens for introgression of new rice for Africa (NERICA) cultivars, which are derivatives of interspecific hybrids between Oryza glaberrima Steud. and O. sativa L. Freshly isolated immature embryos were inoculated with A. tumefaciens LBA4404 that harbored binary vector pBIG-ubi::GUS or pIG121Hm, which each carried a hygromycin-resistance gene and a GUS gene. Growth medium supplemented with 500 mg/l cefotaxime and 20 mg/l hygromycin was suitable for elimination of bacteria and selection of transformed cells. Shoots regenerated from the selected cells on MS medium containing 20 g/l sucrose, 30 g/l sorbitol, 2 g/l casamino acids, 0.25 mg/l naphthaleneacetic acid, 2.5 mg/l kinetin, 250 mg/l cefotaxime, and 20 mg/l hygromycin. The shoots developed roots on hormone-free MS medium containing 30 mg/l hygromycin. Integration and expression of the transgenes were confirmed by PCR, Southern blot analysis, and histochemical GUS assay. Stable integration, expression, inheritance, and segregation of the transgenes were demonstrated by molecular and genetic analyses in the T₀ and T₁ generations. Most plants were normal in morphology and fertile. The transformation protocol produced stable transformants from 16 NERICA cultivars. We also obtained transformed plants by inoculation of calluses derived from mature seeds, but the frequency of transformation was lower and sterility was more frequent.     

Expression of deuterium-isotope-labelled protein in the yeast Pichia pastoris for NMR studies

Deuterium isotope labelling is important for NMR studies of large proteins and complexes. Many eukaryotic proteins are difficult to express in bacteria, but can be efficiently produced in the methylotrophic yeast Pichia pastoris. In order to facilitate NMR studies of the malaria parasite merozoite surface protein-1 (MSP1) complex and its interactions with antibodies, we have investigated production of the MSP1-19 protein in P. pastoris grown in deuterated media. The resulting deuteration patterns were analyzed by NMR and mass spectrometry. We have compared growth characteristics and levels of heterologous protein expression in cells adapted to growth in deuterated media (95% D₂O), compared with expression in non-adapted cells. We have also compared the relative deuteration levels and the distribution pattern of residual protiation in protein from cells grown either in 95% D₂O medium with protiated methanol as carbon source, or in 95% D₂O medium containing deuterated methanol. A high level of uniform C deuteration was demonstrated, and the consequent reduction of backbone amide signal linewidths in [¹H/¹⁵N]-correlation experiments was measured. Residual protiation at different positions in various amino acid residues, including the distribution of methyl isotopomers, was also investigated. The deuteration procedures examined here should facilitate economical expression of ²H/¹³C/¹⁵N-labelled protein samples for NMR studies of the structure and interactions of large proteins and protein complexes.     

Impact of acetaldehyde- and pyruvic acid-bound sulphur dioxide on wine lactic acid bacteria

Aims: To investigate the impact of acetaldehyde‐ and pyruvic acid‐bound sulphur dioxide on wine lactic acid bacteria (LAB). Methods and Results: Growth studies were performed where Oenococcus oeni, Pediococcus parvulus, Ped. damnosus and Lactobacillus hilgardii were inoculated into media containing various concentrations of acetaldehyde or pyruvic acid and an equimolar concentration of SO₂ at pH 3·50 and 3·70. Low concentrations of acetaldehyde‐ and pyruvic acid‐bound SO₂ were inhibitory to the growth of all bacteria although acetaldehyde‐bound SO₂ was generally more inhibitory than pyruvic acid‐bound SO₂. Inhibition was greater at pH 3·50 than 3·70, and Lact. hilgardii was the most sensitive to acetaldehyde‐bound SO₂, while O. oeni was the most sensitive to pyruvic acid‐bound SO₂. Degradation of SO₂‐bound acetaldehyde was observed for all LAB, and aside from O. oeni, there was also complete degradation of SO₂‐bound pyruvic acid at both pH values. O. oeni only degraded pyruvic acid at pH 3·70. Degradation of SO₂‐bound acetaldehyde or pyruvic acid did not correlate with bacterial growth as inhibition was always observed in media containing bound SO₂. Conclusions: Acetaldehyde‐ and pyruvic acid‐bound SO₂ were inhibitory to wine LAB growth at concentrations as low as 5 mg l^?1. Despite this inhibition, all wine LAB degraded SO₂‐bound acetaldehyde and pyruvic acid suggesting that bound SO₂ may have a bacteriostatic rather than bacteriocidal action. Significance and Impact of the Study: Sulphur dioxide bound to acetaldehyde or pyruvic acid is inhibitory to growth of wine LAB and must be considered when conducting the malolactic fermentation or controlling the growth of spoilage bacteria such as Pediococcus and Lactobacillus.     

EFFECTS OF HYBRIDIZATION AND INBREEDING ON FITNESS IN PHLOX

The effects of interpopulation hybridization, and self-fertilization and immigration on fitness in Phlox drummondii were analyzed in 5 natural field sites. Germination rates, survivorship to flowering, fecundity, and net reproductive rates (R_o) were determined for planted populations of natives, hybrids, aliens, and the products of one and two generations of self-fertilization. At all sites, seed germination was 36% for natives, 35% for hybrids and 28% for aliens. Survivorship for natives was 50%, compared to 49% for hybrids and 41% for aliens. The mean fecundity was 34, 35, and 39 seeds per plant for natives, hybrids, and aliens, respectively. The R_o of natives averaged 5.2 vs. 6.4 for hybrids and 4.2 aliens. At all the sites, germination averaged 25% for self-1 plants and 19% for self-2 plants compared to 24% for open-pollinated controls. Survivorship progressed from 50% in the controls to 46% in self-1 and 44% in self-2 plants. Seed production per plant averaged 35% in the control vs. 32% in the self-1 and 25% in the self-2 plants. The mean R_o of the control was 3.8 vs. 3.3 in the self-1 and 2.14 in the self-2 plants. Our results demonstrate that the genetic variable may have a substantial effect on plant fitness in the field.     

Meiotic transmission of T-DNA genes inArabidopsis thaliana plants and their expression after 5-azacytidine treatment

Arabidopsis thaliana tumors were induced by octopine strain ofAgrobacterium tumefaciens B6S3 and its derivatives with modified T-DNA. Flowering shoots appeared sponta-neously onin vitro cultivated tumors and set seeds. R₁ and R₂ progeny of octopine synthesizing plants segregated in opine synthesis activities 3:1 and 15:1. Octopine synthase activity showed absolute linkage with agropine synthesis in most lines. In R₃ and R₄ progenies, the fraction of octopine synthase and agropine synthesis positive plants was lower than expected, but Mendelian segregation was restored if plants were cultivated on medium with 5-azacytidine. The most probable mechanism of disapearance of opine synthesis is cytosine methylation. The effect of 5-azacytidine lasted for at least next two generations.