Adaptation and growth response of Arabidopsis thaliana to deuterium

Summary Arabidopsis seeds were sown aseptically on mineral media containing between 0 and 90% of heavy water (D₂O). Initially, a D₂O level of over 50% was lethal for the plants. However, after culture for six successive generations on 50% D₂O, plants were capable of growing marginally on media containing up to 70% D₂O, but not higher. With increasing concentration, deuterium progressively delays germination, slows growth, reduces survival, results in bleaching of the leaves and delays flowering. Pollen fertility is not affected measurably but seed set is reduced with increasing levels of deuteration so that at 70% D₂O few seeds were obtained. The viability of the seeds harvested from plants grown on deuterated media is low. No chlorophyll or morphological mutants were observed among a large number of plant progenies. Seeds from plants cultured on D₂O media for several generations grow normally on proteated media in the very first generation.     

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.     

Increased hexokinase levels in endosperm of mutant maize

Hexokinase activity was measured in endosperms of shrunken-2 (sh₂) and starchy maize. Initial increases in hexokinase were observed for developing endosperms of both genotypes, and the enzyme declined in both as the seeds matured. A higher level of hexokinase was observed in developing sh₂ than in starchy endosperm. This difference persisted throughout maturation and occurred also in germinating seeds. Soluble hexokinase activity per endosperm continued to increase in sh₂ for about 8 days (22–30 days after pollination) after the enzyme in starchy endosperm had attained maximum activity and begun to decline. Hexokinase was predominantly soluble in both genotypes so the differences observed are not due to altered distribution of enzyme between particulate and soluble fractions.     

Effect of Na+ and K+ Ions on the Initial Crystallization Process of Lysozyme in the Presence of D2O and H2O

In the initial stage of the crystallization of egg-white lysozyme, monomeric lysozyme aggregated rapidly to form a nucleus in the presence of high salt concentrations. In the present studies, we examined the initial aggregation process of lysozyme (initial crystallization process of lysozyme) in D₂O/H₂O with sodium ions or potassium ions, and investigated the relationship between the surface hydrophobicity and the aggregation rate of lysozyme. The effect of sodium ions or potassium ions on the initial aggregation process of lysozyme in D₂O was clearly different from H₂O. The initial aggregation rate of lysozyme in H₂O was slower than in D₂O. In the case of H₂O, the initial aggregation rate was about the same in both ions. But in the case of D₂O, the initial aggregation rate was affected by the ion species and the value was lower in potassium ions than in sodium ions. These results suggest that the interaction between lysozyme molecules is stronger in D₂O than in H₂O. Furthermore, sodium ions have a stronger effect on the interaction than potassium ions in the case of D₂O. There was a good correlation among the initial aggregation rate, surface hydrophobicity, and ζ-potential of lysozyme. The hydrophobic interaction may be an important active force in the initial aggregation process of lysozyme.     

Effect of Na+ and K+ Ions on the Initial Crystallization Process of Lysozyme in the Presence of D2O and H2O

In the initial stage of the crystallization of egg-white lysozyme, monomeric lysozyme aggregated rapidly to form a nucleus in the presence of high salt concentrations. In the present studies, we examined the initial aggregation process of lysozyme (initial crystallization process of lysozyme) in D₂O/H₂O with sodium ions or potassium ions, and investigated the relationship between the surface hydrophobicity and the aggregation rate of lysozyme. The effect of sodium ions or potassium ions on the initial aggregation process of lysozyme in D₂O was clearly different from H₂O. The initial aggregation rate of lysozyme in H₂O was slower than in D₂O. In the case of H₂O, the initial aggregation rate was about the same in both ions. But in the case of D₂O, the initial aggregation rate was affected by the ion species and the value was lower in potassium ions than in sodium ions. These results suggest that the interaction between lysozyme molecules is stronger in D₂O than in H₂O. Furthermore, sodium ions have a stronger effect on the interaction than potassium ions in the case of D₂O. There was a good correlation among the initial aggregation rate, surface hydrophobicity, and -potential of lysozyme. The hydrophobic interaction may be an important active force in the initial aggregation process of lysozyme.     

ATP Production by Respiration and Fermentation, and Energy Charge during Aerobiosis and Anaerobiosis in Twelve Fatty and Starchy Germinating Seeds

The respiration and fermentation rates were compared in germinating seeds of 12 different cultivated species from five families. In air, fermentation contributes significantly to the energy metabolism only in some species (pea, maize), but is generally negligible when compared to respiration. The fermentation rate under anoxia was related either to the metabolic activity under air or to the adenine nucleotide content of the seeds: it was generally higher in seeds which contain starchy reserves (rice, maize, sorghum, pea), than in seeds which do not contain starch (lettuce, sunflower, radish, turnip, cabbage, flax); however, it was similar in wheat, sorghum (starchy seeds), and soya (nonstarchy seeds). The value of the energy charge of all the seeds was lower under anoxia than in air: after 24 hours under anoxia, it was higher than 0.5 in the starchy seeds and in soya and it was around 0.25 in the other fatty seeds.     

Physiological Studies with Heavy Water. II. Germination and Growth Inhibition of Barley by D2O

Seed germination and barley seedling growth in various D₂O concentrations have been studied. It was observed that the emergence of root and shoot was delayed, there being greater delay in shoot than in root emergence. A complete block was observed in germination in pure D₂O and the germination rate was slowed down significantly in lower concentrations. An initial germination delay by different D₂O concentrations seemed to cause a subsequent retardation in the growth measured as shoot and root length. A comparison of root and shoot length with their respective dry weights suggested that the growth by cell division/elongation might have been affected more than the transport of food materials from the endosperm to the embryo. Analysis of the total sugars of the endosperm and the embryo at 8 hour intervals showed that while the hydrolysis of starch to sugars was progressively decreased by increasing D₂O concentrations, the transport rate from endosperm to embryo showed a sharp inhibition in 50% D₂O and above. This indicated that the inhibition in the transport of materials, besides less hydrolysis of reserve food materials, may also be a causal factor of germination and growth inhibition in D₂O.     

Characterization of the increased lysophospholipase activity in gibberellic Acid-treated barley aleurone layers

A lysophospholipase (LPL) activity appears in the aleurone of barley (Hordeum vulgare L. cv Himalaya) half seeds during imbibition on moist agar. Secretion of LPL by half seeds is promoted by GA₃; the increase in secretory rate is almost linear from 10⁻¹⁰ to 10⁻⁶ molar GA₃. LPL activity is likewise promoted in isolated aleurone layers by GA₃. Its secretion into the incubation medium requires the continued presence of GA₃ and commences after a 10 to 14 hour lag period when 10 millimolar Ca²⁺ is present. In the absence of Ca²⁺, the lag period remains unchanged but attainment of the maximum secretory rate is delayed. Ca²⁺ alone has very little effect either on LPL activity accumulated in the aleurone layer or in the surrounding medium. However, 50 millimolar Ca²⁺ together with GA₃ dramatically increase the level of secreted activity and of total (accumulated and secreted) activity.

The metabolic inhibitors cycloheximide and actinomycin D inhibit the accumulation of LPL activity in the aleurone and also the secreted activity. Actinomycin D added after the lag period results in a much lower inhibition. The increase in LPL activity in response to GA₃ occurs as a result of de novo synthesis; LPL activity from barley half seeds incubated in 80% D₂O in the presence of GA₃ undergoes a shift to higher density compared with the activity from similar controls incubated in H₂O. The characteristics of the GA₃ enhancement of LPL activity are compared specifically with α-amylase and generally with other GA₃-controlled hydrolases.

     

Presence of ADP-Glucose Pyrophosphorylase in Shrunken-2 and Brittle-2 Mutants of Maize Endosperm

ADP-Glucose pyrophosphorylase activity has been detected in relatively low amounts in the embryos and endosperms of sh₂ and bt₂ mutant maize seeds. The total enzyme activities in sh₂ and bt₂ were about 12% and 17% respectively, of that found in starchy maize seeds (Dekalb 805). The ADP-glucose pyrophosphorylases from the starchy and mutant maize seeds were activated by 3-phosphoglycerate. However, the extent of the activation of the sh₂ enzyme was not as great as that observed with the bt₂ and Dekalb 805 enzymes. The low levels of ADP-glucose pyrophosphorylase activity in the maize mutants correlate well with the low levels of starch found in the endosperm of these mutants.     

Kinetic Studies on the Initial Crystallization Process of Lysozyme in the Presence of D2O and H2O

In the initial stages of the crystallization of egg-white lysozyme, monomeric lysozyme aggregates rapidly and forms a nucleus in the presence of high salt concentrations. The formation process of the aggregates was examined to make clear the difference between the situations in heavy water and in water at the same sodium ion concentration. The aggregation in both cases was observed at unsaturated and/or saturated lysozyme concentrations. The turbidity at 350 nm of lysozyme increased remarkably within 60 min under each experimental condition and showed no appreciable changes over 60 min. The increase of turbidity in H₂O was much slower than in D₂O at the same salt concentration (3%). Lysozyme showed a critical concentration for nucleus formation whose value in H₂O was lower than in D₂O at 3% salt concentration. There are two different aggregation models, depending on the concentration of lysozyme. However, similar results were not obtained at 3% sodium ions in H₂O. The initial aggregation rate was also dependent on the concentrations of both lysozyme and NaCI. Therefore, the effect of lysozyme concentration on the aggregation process in H₂O may be smaller than in D₂O.     

Protein Synthesis in Dormant and Non-Dormant Cocklebur Seed Segments

Using the axial and cotyledonary segments of lower cocklebur (Xanthium pensylvanicum Wallr.) seeds, protein synthesis as shown by incorporation of radioactive leucine was examined in relation to their dormant status. During the first 9 h of water imbibition, the protein synthesis was higher in the dormant axes than in the non-dormant, after- ripened ones. When imbibed for more than 12 h non-dormant axes had a higher activity than dormant ones. This was also the case with the cotyledonary segments. Cyctoheximide, an inhibitor of protein synthesis, blocked protein synthesis in the axial tissue regardless of its dormant status, and thereby inhibited germination of the non-dormant seeds. In the dormant seeds, however, cycloheximide at 3 mM slightly stimulated germination without stimulating the C₂H₄ production. Based on these results, it is suggested that in cocklebur seeds there may be some proteinaceous system which is involved in the maintenance of dormancy.     

Enhancement by gibberellic acid and asymmetric distribution of lysophospholipase in germinating barley

Germination of whole barley seeds for 4 and 6 days followed by measurement of lysophospholipase (lysolecithin acyl hydrolase, LAH) in the embryo-containing and embryo-free halves revealed a gradient of activity between the two halves of the seed. Most of the activity appeared in the embryo-containing half. This gradient decreased slightly in the aleurone and dramatically in the starchy endosperm during the 2 day germination interval. Embryo-containing and embryo-free half seeds of surface sterilized barley were placed separately on sterile agar plates. After 4 and 6 days LAH was observed in both the aleurone and starchy endosperm of the embryo-containing halves. In the embryo-free halves, LAH appeared at low levels in the aleurone and was virtually absent in the starchy endosperm. The scutellum of germinating seeds contains LAH activity. Exposure of embryo-free half seeds to GA₃ for 24 hr showed enhancement of acidic and alkaline LAH activities in the aleurone fraction and in the GA₃-medium in which the half seeds were treated. The LAH activity of the starchy endosperm of these half seeds was little changed by GA₃ treatment. Exposure of isolated aleurones to GA₃ for 24 hr resulted in substantial enhancement of acidic and alkaline LAH activities in the bathing medium and in fractions prepared from the aleurone. The physiological significance of the influence of GA₃ on LAH activity during barley germination is discussed.     

Uptake of Gibberellic Acid in Intact and Scarified Seeds of Barbarea vulgaris

Uptake of gibberellic acid as a function of duration of exposure, external concentration, and seed lot was measured in seeds of yellow rocket (Barbarea vulgaris R. Br.) by means of lettuce hypocotyl bioassays of the acidic, basic, or neutral fractions of seed extracts and by uptake of ¹⁴C-GA₃. In intact seeds, where mM levels of GA₃ promoted only 25% germination, uptake was completed within 24 h of exposure. The maximum uptake was about 0.2% of external amount. Although germination promotion by GA₃ differed among seedlots of yellow rocket, relative uptake (percentage of the external GA₃) was nearly the same. The relative rate of uptake of GA₃ was similar for scarified and intact seeds, but germination was promoted in scarified seeds by much lower levels of GA₃ than in intact seeds. Total uptake in scarified seeds was much higher, however (about 10% of the external amount). In seeds imbibed in H₂O, practically no endogenous GA-like activity was detected in either the acidic, basic, or neutral fractions. It was also apparent that intact seeds could take up quantities of GA₃ that failed to promote germination, but were comparable to quantities that promoted germination in scarified seeds.     

Kinetic Studies on the Initial Crystallization Process of Lysozyme in the Presence of D2O and H2O

In the initial stages of the crystallization of egg-white lysozyme, monomeric lysozyme aggregates rapidly and forms a nucleus in the presence of high salt concentrations. The formation process of the aggregates was examined to make clear the difference between the situations in heavy water and in water at the same sodium ion concentration. The aggregation in both cases was observed at unsaturated and/or saturated lysozyme concentrations. The turbidity at 350 nm of lysozyme increased remarkably within 60 min under each experimental condition and showed no appreciable changes over 60 min. The increase of turbidity in H₂O was much slower than in D₂O at the same salt concentration (3%). Lysozyme showed a critical concentration for nucleus formation whose value in H₂O was lower than in D₂O at 3% salt concentration. There are two different aggregation models, depending on the concentration of lysozyme. However, similar results were not obtained at 3% sodium ions in H₂O. The initial aggregation rate was also dependent on the concentrations of both lysozyme and NaCI. Therefore, the effect of lysozyme concentration on the aggregation process in H₂O may be smaller than in D₂O.     

Shaker-IR K+ channel gating in heavy water: Role of structural water molecules in inactivation

It has been reported earlier that the slow (C-type) inactivated conformation in K_v channels is stabilized by a multipoint hydrogen-bond network behind the selectivity filter. Furthermore, MD simulations revealed that structural water molecules are also involved in the formation of this network locking the selectivity filter in its inactive conformation. We found that the application of an extracellular, but not intracellular, solution based on heavy water (D₂O) dramatically slowed entry into the slow inactivated state in Shaker-IR mutants (T449A, T449A/I470A, and T449K/I470C, displaying a wide range of inactivation kinetics), consistent with the proposed effect of the dynamics of structural water molecules on the conformational stability of the selectivity filter. Alternative hypotheses capable of explaining the observed effects of D₂O were examined. Increased viscosity of the external solution mimicked by the addition of glycerol had a negligible effect on the rate of inactivation. In addition, the inactivation time constants of K⁺ currents in the outward and the inward directions in asymmetric solutions were not affected by a H₂O/D₂O exchange, negating an indirect effect of D₂O on the rate of K⁺ rehydration. The elimination of the nonspecific effects of D₂O on our macroscopic current measurements supports the hypothesis that the rate of structural water exchange at the region behind the selectivity filter determines the rate of slow inactivation, as proposed by molecular modeling.     

Nuclear Magnetic Resonance Evidence using D2O for Structured Water in Muscle and Brain

The electric quadrupole moment of the deuterium nucleus provides a nuclear magnetic resonance (NMR) probe of electric field gradients, and thereby of organization of tissue water. 8-17% of H₂O in rat muscle and brain was replaced by D₂O from 50% deuterated drinking water. The peak height of the steady-state NMR spectrum of D in muscle water was 74% lower than that of an equal concentration of D₂O in liquid water. Longitudinal NMR relaxation times (T₁) of D in water of muscle and brain averaged 0.092 and 0.131 sec, respectively, compared with 0.47 sec in D₂O in liquid water. Transverse NMR relaxation times (T₂) averaged 0.009 and 0.022 sec in D₂O of muscle and brain, respectively, compared with 0.45 sec in D₂O in liquid water. These differences cannot be explained by paramagnetic ions or by magnetic inhomogeneities, which leaves increased organization of tissue water as the only tenable hypothesis. Evidence was also obtained that 27% of muscle water and 13% of brain water exist as a separate fraction with T₂ of D₂O less than 2 × 10^-3 sec, which implies an even higher degree of structure. Each of the two fractions may consist of multiple subfractions of differing structure.     

Solvent isotope effects on microtubule polymerization and depolymerization

The initial velocity of polymerization of purified beef brain tubulin has been determined at various values of pH or pD in water and in H₂O-D₂O mixtures. D₂O was shown to inhibit both polymerization at 37 °C and depolymerization measured at 5 °C and 37 °C. The microtubules formed in D₂O were indistinguishable from those formed in H₂O, by electron microscope examination. In 93% D₂O the pL²versus rate of polymerization curve was displaced about one unit towards higher pL values. In certain regions of the pL versus rate curve, a stimulation in the rate of polymerization by D₂O is observed. The extent of polymerization at the optimum pL value was not affected by D₂O.     

A vibrational spectroscopic study of the ice-melting-induced transition of 1,2-dibehenoyl-sn-glycero-3-phosphocholine

The phase transition of bilayers of 1,2-dibehenoyl-sn-glycero-3-phosphocholine (DBPC) induced by ice (H₂O and D₂O) melting has been investigated by infrared and Raman spectroscopy. Spectral changes observed at this transition are smaller at lower water content. These spectral changes are interpreted in terms of increased molecular mobility. Slightly different temperature dependencies are observed for various spectral parameters between samples dispersed in H₂O and D₂O.     

Effect of heavy water on the germination of a number of species of seeds

Summary The effect of deuteriation on the germination rate of seeds is studied as a possible screening technique prior to the cultivation of a plant in high concentrations of D₂O. There appears to be a simple relationship between size of the seed and germination capacity in high deuterium concentrations. Larger seeds may be more successful in germinating because of greater hydrogen-containg food reserves.