Induction of secondary dormancy in sunflower seeds by high temperature. Possible involvement of ethylene biosynthesis

High temperature (45°C) inhibits seed germinition and seedling sunflower ( Helianthus annuus L. cv. Mirasol). Treatment of imbibed seeds at 45°C for more than 48 h induces a secondary dormancy, which is associated with progressive decrease of germination ability at optimal temperature (25°C) as well as with abnormal seedling growth. Ethylene (55μl l⁻¹) and 2-chloroethylphosphonic acid (ethephon) (2.5 m M ) improve germination of thermodormant seeds at 25°C. but the abnormal growth of the seedlings remains. O₂-enriched atmosphere and dry storage improve germination and normal seedling growth. The induction of thermodormancy in sunflower seeds seems associated with loss of their ability to convert 1-aminocyclopropane-1-carboxylic acid (ACC) to ethylene. Possible effects of high temperature on membranes and ethylene forming enzyme (EFE) are discussed.     

A rapid non-destructive assay to quantify soybean nodule gas permeability

The low gas permeability of a diffusion barrier in the cortex of soybean nodules plays a significant role in the protection of nitrogenase from oxygen inactivation. It may also set an upper limit on nodule respiration and nitrogen fixation rates. Two methods which have been used to quantify the gas permeability of leguminous nodules are reviewed and found to be unreliable. A new assay technique for determining both the nodule activity and gas permeability is developed and tested. This ‘lag-phase’ assay is based on the time nodules require to reach steady-state ethylene production after being exposed to acetylene. The technique is rapid, insensitive to errors in biochemical parameters associated with nitrogenase, and is non-destructive. The method was tested with intact aeroponically grown soybean plants for which the mean nodule gas permeability was found to be 13.3×10⁻³ mms⁻¹. This corresponds to a layer of cells approximately 35 um thick and is consistent with previously reported values.     

Soybean nodule gas permeability,nitrogen fixation and dirunal cycles in soil temperature

While diurnal cycles in nitrogen fixation rates are sometimes assumed to result from diurnal variation in photosynthetically active radiation, contradicting evidence exists that indicate soil temperature is the primary environmental influence. These studies assessed the significance of temperature on soybean nitrogen fixation under field conditions. Two groups of intact field-grown soybean plants, one at ambient and the other exposed to a 10°C diurnal variation in soil temperature, were nondestructively assayed for acetylene reduction rates. Activity was closely associated with soil temperature (R²=0.85), even when temperature was 12 h out of phase with ambient. Data were also obtained to determine if the effects of rhizosphere temperature on nitrogen fixation are mediated through an effect on the nodule oxygen permeability. Nodule oxygen permeability of intact, aeroponically grown soybean was closely correlated with the diurnal changes in temperature (R²=0.90).     

Heterotrophic metabolism and diazotrophic growth ofNostoc sp. fromCycas circinalis

The cyanobiont ofCycas circinalis (identified asNostoc sp.) was isolated and its heterotrophic metabolism was studied in free culture under nitrogen-fixing conditions. Morphology, growth rate, nitrogenase activity, biochemical composition, efficiency of assimilation of organic carbon and molecular nitrogen were determined under different conditions of energy and carbon supply. The study has revealed the high potential of the heterotrophic metabolism in this symbiotic cyanobacterium. Although low rates of metabolic activities were attained under heterotrophic conditions, the efficiencies of organic carbon utilization (0.48 g cell-carbon per g glucose-carbon in chemoheterotrophy, from 0.65 to 0.74 under photoheterotrophy) and of N₂ assimilation (35.0 mg N₂ fixed per g glucose used in chemoheterotrophy, from 58.3 to 61.9 under photoheterotrophy) displayed by this organism were among the highest ever found in diazotrophically grown microorganisms. The isolate fromC. circinalis was able to grow indefinitely in the dark under nitrogen-fixing conditions, maintaining a well balanced biosynthetic activity and the capacity to resume photosynthetic metabolism quickly. The significance of the heterotrophic potential of this symbioticNostoc is discussed.     

Temperature effects on malic-acid efflux from the vacuoles and on the carboxylation pathways in crassulacean-acid-metabolism plants

The studies described in the paper were conducted with tissue slices of Crassulacean acid metabolism (CAM) plants floating in isotonic buffer. In a first series of experiments, temperature effects on the efflux of [¹⁴C]malate and¹⁴CO₂ were studied. An increase of temperature increased the efflux from the tissue in a non-linear manner. The efflux was markedly influenced also by the temperatures applied during the pretreatment. The rates of label export in response to the temperature and the relative contributions of¹⁴CO₂ and [¹⁴C]malate to the label export were different in the two studied CAM plants (Kalanchoë daigremontiana, Sempervivum montanum). In further experiments, temperature response of the labelling patterns produced by¹⁴CO₂ fixation and light and darkness were studied. In tissue which had accumulated malate (acidified state) an increase of temperature decreased the rates of dark CO₂ fixation whilst the rates of CO₂ fixation in light remained largely unaffected. An increase of temperature shifted the labelling patterns from a C₄-type (malate being the mainly labelled compound) into a C₃-type (label in carbohydrates). No such shift in the labelling patterns could be observed in the tissue which had depleted the previously stored malate (deacidified state). The results indicate that in the acidified tissue the increase of temperature increases the efflux of malate from the vacuole by changing the properties of the tonoplast. It is assumed that the increased export of malic acid lowers the in-vivo activity of phosphoenol pyruvate carboxylase by feedback inhibition.Abbreviations CAM Crassulacean acid metabolism - FW fresh weight - PEPCase phosphoenolpyruvate carboxylase Dedicated to Professor O.L. Lange, Würzburg, on the occasion of his 60th birthday     

The effect of auxin concentration on cytokinin stability and metabolism

The stability of [³H]zeatin riboside supplied to freshly excised tobacco pith explants was found to be inversely related to -naphthaleneacetic acid concentration in the incubation medium. At higher concentrations of -naphthaleneacetic acid greater breakdown of [³H]zeatin riboside was indicated by higher levels of degradative metabolites (adenine, adenosine and adenosine nucleotides) formed. This auxin effect on cytokinin metabolism appears to be mediated, at least in part, through cytokinin oxidase. The results of in-vitro assays carried out with partially purified enzyme from corn kernels substantiale this conclusion. These findings are discussed in relation to recent observations of auxin and cytokinin levels in crown-gall tumours with altered morphology.Abbreviations FPLC fast protein liquid chromatography - HPLC high-performance liquid chromatography - IP isopentenyladenine - NAA naphthaleneacetic acid - ZR zeatin riboside     

Phosphate sequestration by glycerol and its effects on photosynthetic carbon assimilation by leaves

Glycerol induced a limitation on photosynthetic carbon assimilation by phosphate when supplied to leaves of barley (Hordeum vulgare L.) and spinach (Spinacia oleracea L.). This limitation by phosphate was evidenced by (i) reversibility of the inhibition of photosynthesis by glycerol by feeding orthophosphate (ii) a decrease in light-saturated rates of photosynthesis and saturation at a lower irradiance, (iii) the promotion of oscillations in photosynthetic CO₂ assimilation and in chlorophyll fluorescence, (iv) decreases in the pools of hexose monophosphates and triose phosphates and increases in the ratio of glycerate-3-phosphate to triose phosphate, (v) decreased photochemical quenching of chlorophyll fluorescence, and increased non-photochemical quenching, specifically of the component which relaxed rapidly, indicating that thylakoid energisation had increased. In barley there was a massive accumulation of glycerol-3-phosphate and an increase in the period of the oscillations, but in spinach the accumulation of glycerol-3-phosphate was comparatively slight. The mechanism(s) by which glycerol feeding affects photosynthetic carbon assimilation are discussed in the light of these results.Abbreviations Chl chlorophyll - C _i intercellular concentration of CO₂ - P phosphate - PGA glycerate-3-phosphate - Pi orthophosphate - triose-P sum of glyceraldehyde-3-phosphate and dihydroxyacetone phosphate     

Regulation of malic-acid metabolism in Crassulacean-acid-metabolism plants in the dark and light: In-vivo evidence from 13C-labeling patterns after 13CO2 fixation

The labeling patterns in malic acid from dark ¹³CO₂ fixation in seven species of succulent plants with Crassulacean acid metabolism were analysed by gas chromatography-mass spectrometry and ¹³C-nuclear magnetic resonance spectrometry. Only singly labeled malic-acid molecules were detected and on the average, after 12–14 h dark ¹³CO₂ fixation the ratio of [4-¹³C] to [1-¹³C] label was 2:1. However the 4-C carboxyl contained from 72 to 50% of the label depending on species and temperature. The ¹³C enrichment of malate and fumarate was similar. These data confirm those of W. Cockburn and A. McAuley (1975, Plant Physiol. 55, 87–89) and indicate fumarase randomization is responsible for movement of label to 1-C malic acid following carboxylation of phosphoenolpyruvate. The extent of randomization may depend on time and on the balance of malic-acid fluxes between mitochondria and vacuoles. The ratio of labeling in 4-C to 1-C of malic acid which accumulated following ¹³CO₂ fixation in the dark did not change during deacidification in the light and no doubly-labeled molecules of malic acid were detected. These results indicate that further fumarase randomization does not occur in the light, and futile cycling of decarboxylation products of [¹³C] malic acid (¹³CO₂ or [1-¹³C]pyruvate) through phosphoenolpyruvate carboxylase does not occur, presumably because malic acid inhibits this enzyme in the light in vivo. Short-term exposure to ¹³CO₂ in the light after deacidification leads to the synthesis of singly and multiply labeled malic acid in these species, as observed by E.W. Ritz et al. (1986, Planta 167, 284–291). In the shortest times, only singly-labeled [4-¹³C]malate was detected but this may be a consequence of the higher intensity and better detection statistics of this ion cluster during mass spectrometry. We conclude that both phosphoenolpyruvate carboxylase (EC 4.1.1.32) and ribulose-1,5-biphosphate carboxylase (EC 4.1.1.39) are active at this time.Abbreviations CAM Crassulacean acid metabolism - GCMS gas chromatography-mass spectrometry - MS mass spectrometry - NMR nuclear magnetic resonance spectrometry - PEP phosphoenolpyruvate - RuBP ribulose 1,5-bisphosphate     

Plasmalemma- and tonoplast-ATPase activity in mesophyll protoplasts,vacuoles and microsomes of the Crassulacean-acid-metabolism plant Kalanchoe daigremontiana

Adenosine-triphosphatase activity on the plasmalemma and tonoplast of isolated mesophyll protoplasts, isolated vacuoles and tonoplast-derived microsomes of the Crassulacean-acid-metabolism plant Kalanchoe daigremontiana Hamet et Perr., was localized by a cytochemical procedure using lead citrate. Enzyme activity was detected on the cytoplasmic surfaces of the plasmalemma and tonoplast. The identity of the enzymes was confirmed by various treatments differentiating the enzymes by their sensitivity to inhibitors of plasmalemma and tonoplast H⁺-ATPase. Isolated vacuoles and microsomes prepared from isolated vacuoles clearly exhibited single-sided deposition on membrane surfaces.Abbveviations CAM Crassulacean acid metabolism - H⁺-ATPase proton-translocating ATPase     

Human hepatocyte and kidney cell metabolism of 2-acetylaminofluorene and comparison to the respective rat cells

The metabolism and mutagenic activation of 2-acetylaminofluorene by human and rat hepatocytes and kidney cells were measured. High performance liquid chromatography was used to separate the 2-acetylaminofluorene metabolites, and a cell-mediated Salmonella typhimurium mutagenesis assay was used to detect mutagenic intermediates. Rat and human differences were observed with cells from both organs and levels of metabolism and mutagenesis were higher in human cells. Within a species, liver and kidney cell differences were also evident, with levels of hepatocyte-mediated metabolism and mutagenesis being greater than kidney cells. Human inter-individual variation was apparent with cells from both organs, but the variation observed was significantly greater in hepatocytes than kidney cells. A knowledge of such differences, including an understanding that they may vary with the chemical being studied, should be useful in the extrapolation of rodent carcinogenesis data to humans.Abbreviations AAF 2-acetylaminofluorene - AF 2-aminofluorene - DMSO dimethylsulfoxide - HPLC high performance liquid chromatography - N-OH-AAF N-hydroxy-2-acetylaminofluorene - 1-OH-AAF 1-hydroxy-2-acetylaminofluorene - 3-OH-AAF 3-hydroxy-2-acetylaminofluorene - 5/9-OH-AAF a combination of 5 and 9-hydroxy-2-acetylaminofluorene - 7-OH-AAF 7-hydroxy-2-acetylaminofluorene - 8-OH-AAF 8-hydroxy-2-acetylaminofluorene