Comprehensive RNAA of cadmium,cobalt, nickel,and copper using109Cd,57Co,and reactor-produced67Cu as radioisotopic yield monitors

An existing radiochemical NAA procedure for Cd, Co, and Cu was improved to allow determination of individual radiochemical yields by the radioisotopic tracer technique, thus eliminating errors owing to variable recovery.¹⁰⁹Cd was used as tracer for Cd determination via¹¹⁵Cd/^115mIn,⁵⁷Co for Co via⁶⁰Co, and potentially for Ni via⁵⁸Co, whereas as a novelty⁶⁷Cu, produced by reactor irradiation of ZnO of natural isotopic composition (by the⁶⁷Zn [n,p]⁶⁷Cu reaction) was used for Cu via the indicator nuclide⁶⁴Cu. The simple production and purification of⁶⁷Cu by anion exchange is described. Results for biological RMs are given and discussed.     

Vanadate inhibition of the Ca-ATPase from human red cell membranes

1. (1) VO₃⁻ combines with high affinity to the Ca²⁺-ATPase and fully inhibits Ca²⁺-ATPase and Ca²⁺-phosphatase activities. Inhibition is associated with a parallel decrease in the steady-state level of the Ca²⁺-dependent phosphoenzyme.

2. (2) VO₃⁻ blocks hydrolysis of ATP at the catalytic site. The sites for VO₃⁻ also exhibit negative interactions in affinity with the regulatory sites for ATP of the Ca²⁺-ATPase.

3. (3) The sites for VO₃⁻ show positive interactions in affinity with sites for Mg²⁺ and K⁺. This accounts for the dependence on Mg²⁺ and K⁺ of the inhibition by VO₃⁻. Although, with less effectiveness, Na⁺ substitutes for K⁺ whereas Li⁺ does not. The apparent affinities for Mg²⁺ and K⁺ for inhibition by VO₃⁻ seem to be less than those for activation of the Ca²⁺-ATPase.

4. (4) Inhibition by VO₃⁻ is independent of Ca²⁺ at concentrations up to 50 μM. Higher concentrations of Ca²⁺ lead to a progressive release of the inhibitory effect of VO₃⁻.

Enzymatic studies on the interaction of myosin and heavy meromyosin with 1,N 6 -ethenoadenosine triphosphate ( ATP), a fluorescent analog of ATP

The fluorescent analog of adenosine triphosphate (ATP)¹ 1,N⁶-ethenoadenosine triphosphate, (εATP), has been utilized as a substitute for ATP in the myosin and heavy meromyosin ATPase systems. For myosin, the analog εATP replaced ATP with a somewhat larger K_m (2.6 × 10^?4 mole ?^?1 for εATP as opposed to 8.8 × 10^?5 mole ?^?1 for ATP), indicating that the apparent affinity of the enzyme for εATP is less than for ATP. Perhaps of more interest, further comparison yielded a V_max for εATP about two and one half times the value for ATP (20 μmole PO₄ sec^?1 g protein^?1 as opposed to 8.1 μmole sec^?1 g protein^?1). Results for the HMM-εATPase system were similar, yielding a K_m value of 1.47 × 10^?4 mole ?^?1 and a V_max of 54.2 μmole PO₄ sec^?1 g protein^?1, as opposed to corresponding K_m and V_max values of 1.23 × 10^?4 mole ?^?1 and 20.4 μmole PO₄ sec^?1 g protein^?1, respectively for the HMM-ATP interaction. The pH dependence of εATPase for both systems was comparable to ATP, suggesting a similarity in the mechanism of hydrolysis of the two nucleotides. Activation of εATPase by Ca²⁺ in the presence of 0.5 M KCl was comparable to ATPase for both systems, but inhibition by Mg²⁺ seemed to be more effective for εATPase. These results indicate that εATP is an excellent substitute for ATP in the myosin and heavy meromyosin systems and because of its insertion into the active site of these muscle proteins, it promises to be a very useful probe for conformation studies at this level.     

The alcohol dehydrogenase alleloenzymes AdhS and AdhF from the fruitfly Drosophila melanogaster: An enzymatic rate assay to determine the active-site concentration

A rapid and reproducible enzymatic rate assay for the quantitative determination of the concentration of active sites is presented for the alleloenzymes Adh^S and Adh^F from Drosophila melanogaster. Using this procedure the turnover numbers as catalytic-center activities were found to be 12.2 sec^–1 for Adh^F and 3.4 sec^–1 for Adh^S with secondary alcohols. This showed a slower dissociation of the coenzyme from the binary enzyme-NADH complex with Adh^S and hence a stronger binding of NADH to this alleloenzyme. With ethanol, the catalytic-center activity was 1.4 sec^–1 for Adh^S and 2.8 sec^–1 for Adh^F, and hence the single amino acid mutation distinguishing the two alleloenzymes also affected hydride transfer.     

K+/H+ antiport in mitochondria

Mitochondria contain a latent K⁺/H⁺ antiporter that is activated by Mg²⁺-depletion and shows optimal activity in alkaline, hypotonic suspending media. This K⁺/H⁺ antiport activity appears responsible for a respiration-dependent extrusion of endogenous K⁺, for passive swelling in K⁺ acetate and other media, for a passive exchange of matrix⁴²K⁺ against external K⁺, Na⁺, or Li⁺, and for the respiration-dependent ion extrusion and osmotic contraction of mitochondria swollen passively in K⁺ nitrate. K⁺/H⁺ antiport is inhibited by quinine and by dicyclohexylcarbodiimide when this reagent is reacted with Mg²⁺-depleted mitochondria. There is good suggestive evidence that the K⁺/H⁺ antiport may serve as the endogenous K⁺-extruding device of the mitochondrion. There is also considerable experimental support for the concept that the K⁺/H⁺ antiport is regulated to prevent futile influx-efflux cycling of K⁺. However, it is not yet clear whether such regulation depends on matrix free Mg²⁺, on membrane conformational changes, or other as yet unknown factors.     

Additivity-based design of the strongest possible turkey ovomucoid third domain inhibitors for porcine pancreatic elastase (PPE) and Streptomyces griseus protease B (SGPB)

We describe here successful designs of strong inhibitors for porcine pancreatic elastase (PPE) and Streptomyces griseus protease B (SGPB). For each enzyme two inhibitor variants were designed. In one, the reactive site residue (position 18) was retained and the best residues were substituted at contact positions 13, 14, and 15. In the other variant the best residues were substituted at all contact positions except the reactive site where a Gly was substituted. The four designed variants were: for PPE, T¹³E¹⁴Y¹⁵-OMTKY3 and T¹³E¹⁴Y¹⁵G¹⁸M²¹P³²V³⁶-OMTKY3, and for SGPB, S¹³D¹⁴Y¹⁵-OMTKY3 and S¹³D¹⁴Y¹⁵G¹⁸I¹⁹K²¹-OMTKY3. The free energies of association (ΔG⁰) of expressed variants have been measured with the proteases for which they were designed as well as with five other serine proteases and the results are discussed.     

Evidence for cooperative effects in the binding of polyvalent metal ions to pure phosphatidylcholine bilayer vesicle surfaces

An internal NMR monitor for the study of lanthanide ion (Ln³⁺) binding to phospholipid bilayer membranes has been developed. The dimethylphosphate anion, DMP^?, forms labile complexes with Ln³⁺ in aqueous solution and in solutions also containing bilayer dispersions. The hyperfine shift in the DMP^? resonance induced by Pr³⁺ ions has been used to determine the overall thermodynamic formation constants for the Pr(DMP)²⁺ and Pr(DMP)₂⁺ complexes: 81 (M^?1) and 349 (M^?2) at 52°C; the limiting hyperfine shift (³¹P) at 52°C is 91.5 ppm downfield. These parameters, applied to the observed DMP^? hyperfine shift in the presence of the membrane, establish both the free Pr³⁺ concentration and the amount of Pr³⁺ bound to the phospholipid surface. Extensive data for the binding of Pr³⁺ to the outer surfaces of sonicated vesicles yield a limiting hyperfine shift per Pr³⁺ of 181.6 ppm downfield for the dipalmitoylphosphatidylcholine ³¹P resonance at 52°C, clearly demonstrating that the binding stoichiometry is two DPPCs per Pr³⁺. A Hill analysis indicates that the binding data are more anti-cooperative than a realistic Langmuir isotherm, yet more cooperative than a Stern isotherm incorporating electrostatic considerations at the Debye-Hückel level. Fittings to specific models lead to a cooperative model in which tense (T) sites, with low affinity for Pr³⁺, present in the absence of metal ions, quickly give way to relaxed (R) sites (two DPPCs per site), with much higher affinity for Pr³⁺, as the amount of Pr³⁺ bound to the surface increases. The intrinsic equilibrium constants for the binding of Pr³⁺ to DPPC vesicles are 2 M^?1 and 3 000 M^?1 for the T and R sites, respectively, at 52°C. The distribution coefficient between these sites ([R]/[T]) in the absence of Ln³⁺ is 0.14 at 52°C. We picture the binding site conversion as a head-group conformational change involving mostly the choline moiety. Sketchy results for binding on the inside vesicle surface indicate that the overall affinity for Pr³⁺ is significantly greater and suggest that the site stoichiometry may be different.     

Substrate kinetics of the tonoplast h-translocating inorganic pyrophosphatase and its activation by free mg

To clarify the kinetic characteristics and ionic requirements of the tonoplast H⁺-translocating inorganic pyrophosphatase (H⁺-PPiase), PPi hydrolysis and PPi-dependent H⁺ transport were studied in tonoplast vesicles isolated from leaf mesophyll tissue of Kalanchoë daigremontiana Hamet et Perrier de la Bâthie. The tonoplast H⁺-PPiase showed an absolute requirement for a monovalent cation and exhibited hyperbolic kinetics with respect to cation concentration. H⁺-PPiase activity was maximal in the presence of K⁺ (K₅₀ approximately 3 millimolar), with PPi-dependent H⁺ transport being more selective for K⁺ than PPi hydrolysis. When assayed in the presence of 50 millimolar KCl at fixed PPi concentrations, H⁺-PPiase activity showed sigmoidal kinetics with respect to total Mg concentration, reflecting a requirement for a Mg/PPi complex as substrate and free Mg²⁺ for activation. At saturating concentrations of free Mg²⁺, H⁺-PPiase activity exhibited Michaelis-Menten kinetics towards MgPPi²⁻ but not Mg₂PPi, demonstrating that MgPPi²⁻ was the true substrate of the enzyme. The apparent K_m (MgPPi²⁻) for PPi hydrolysis (17 micromolar) was significantly higher than that for PPi-dependent H⁺ transport (7 micromolar). Free Mg²⁺ was shown to be an allosteric activator of the H⁺-PPiase, with Hill coefficients of 2.5 for PPi hydrolysis and 2.7 for PPi-dependent H⁺ transport. Half-maximal H⁺-PPiase activity occurred at a free Mg²⁺ concentration of 1.1 millimolar, which lies within the range of accepted values for cytosolic Mg²⁺. In contrast, cytosolic concentrations of K⁺ and MgPPi²⁻ appear to be saturating for H⁺-PPiase activity. We propose that one function of the H⁺-PPiase may be to act as an ancillary enzyme that maintains the proton-motive force across the vacuolar membrane when the activity of the tonoplast H⁺-ATPase is restricted by substrate availability. As ATP levels decline in the cytosol, free Mg²⁺ would be released from the MgATP²⁻ complex, thereby activating the tonoplast H⁺-PPiase.     

The effect of temperature on viability of imbibed weed seeds

Imbibed seed of 10 common arable weeds were placed in trays in initially moist soil and, after imbibing for 2h, heated in ovens/incubators set to 31^oC, 42^oC, 56^oC, 75^oC or 100^oC for 0.5, 1, 2, 4, 8 or 16 days or at 102^oC, 155^oC, 204^oC or 262^oC for 0.5, 1, 2, 5, 7.5 or 10 min. After heating, seeds were incubated for 28 days at 10/20^oC or 20/30^oC on a 12 h dark/light regime, depending on species, and germination recorded. At the lower temperatures, germination of all species was prevented by temperatures of 75^oC or higher for periods of 0.5 days or more. Germination was lower after treatment at 56^oC than at 31^oC or 42^oC for all species except Rumex obtusifolius. The maximum temperature required to prevent germination varied among species and was of greater importance than the duration of heating. Germination was variable with duration of heating. At the higher temperatures, there was very little germination of any species after heating at 204^oC for 7.5 min or 262^oC for 5 min or more. Seeds were greatly buffered from the air temperature by 3 mm of soil, throughout the shorter duration of heating. The average temperature of the soil, over the 10 min heating required to prevent over 90% germination, varied among species and ranged from 48^oC for Avena fatua to 65^oC for R. obtusifolius. This work implies that composting systems maintained at 65^oC are unlikely to provide an efficient method of weed control. Recommendations for improvement of the laboratory technique are suggested.     

Mechanisms by which Li+ stimulates the (Na++K+)-dependent ATPase

The addition of LiCl stimulated the (Na⁺+K⁺)-dependent ATPase activity of a rat brain enzyme preparation. Stimulation was greatest in high Na⁺/low K⁺ media and at low Mg. ATP concentrations. Apparent affinities for Li⁺ were estimated at the α-sites (moderate-affinity sites for K⁺ demonstrable in terms of activation of the associated K⁺-dependent phosphatase reaction), at the β-sites (high-affinity sites for K⁺ demonstrable in terms of activation of the overall ATPase reaction), and at the Na⁺ sites for activation. The relative efficacy of Li⁺ was estimated in terms of the apparent maximal velocity of the phosphatase and ATPase reactions when Li⁺ was substituted for K⁺, and also in terms of the relative effect of Li⁺ on the apparent K_M for Mg· ATP. With these data, and previously determined values for the apparent affinities of K⁺ and Na⁺ at these same sites, quantitative kinetic models for the stimulation were examined. A composite model is required in which Li⁺ stimulates by relieving inhibition due to K⁺ and Na⁺ (i) by competing with K⁺ for the α-sites on the enzyme through which K⁺ decreases the apparent affinity for Mg·ATP and (ii) by competing with Na⁺ at low-affinity inhibitory sites, which may represent the external sites at which Na⁺ is discharged by the membrane NA⁺/K⁺ pump that this enzyme represents. Both these sites of action for Li⁺ would thus lie, in vivo, on the cell exterior.     

Sorption of Heavy Metal Cations by Corn Mitochondria and the Effects on Electron and Energy Transfer Reactions

The passive sorption of Pb⁺², Cd⁺², Zn⁺², Co⁺², Ni⁺², and Mn⁺² by isolated corn mitochondria was determined, and, except for Pb⁺², the maximum sorption for each cation was about 58 nmol per milligram of protein. Sorption of Pb⁺² was apparently ten times greater, but precipitation may have been the cause of this larger value. The effects of Pb⁺², Cd⁺², Zn⁺², Co⁺², and Ni⁺² on acceptorless rates of electron transport for three substrates were determined. Greater than 50% inhibitions of oxidation were observed for succinate after additions of >0.1 mM Cd⁺², Zn⁺², or Pb⁺²: for NADH after additions of >0.5 mM Cd⁺² or Zn⁺²; and for malate + pyruvate after additions of >0.1 mM Cd⁺². Some inhibition of the rate of substrate oxidation was observed for most cations at higher concentrations. Coupling, as measured by ADP/O ratios, was inhibited at lowest concentrations by Cd⁺² or Zn⁺² and at higher concentrations by Co⁺² or Ni⁺². Substantial swelling of mitochondria oxidizing succinate was observed following additions of O.1 mM Cd⁺² or Pb⁺², Correlations are drawn between the effects of Pb⁺², Cd⁺², Zn⁺², Co⁺², and Ni⁺² and their sorption to mitochondrial membranes.     

Surface potential of phosphatidylserine monolayers II. Divalent and monovalent ion binding

Ion binding constants for phosphatidylserine membranes have been derived from the variation of the surface potential of phosphatidylserine monolayers with divalent cation concentrations in the presence of various monovalent salts in the aqueous subphase. The observed surface potential data for the monolayers, analyzed by use of the Gouy-Chapman diffuse potential theory, together with a simple binding reaction formula, yield, for Ca²⁺, Mg²⁺, Na⁺ and (Me)₄N⁺ binding constant values of 30 M^?1, 10 M^?1, 0.6 M^?1 and 0.05 M^?1, respectively. The effect of pH on surface potential of phosphatidylserine monolayers was found to be dependent upon ionic species other than H⁺ in the subphase solution. The distinction between apparent and intrinsic dissociation constants of H⁺ for biomolecules was made in terms of ion binding due to other ions at the same site as for H⁺ in biomolecules.     

Interactions ofRhizobium japonicum strains in soybean nodules and their contribution to nitrogen fixation

Summary Rhizobium japonicum strain 8-0 Str^R applied as inoculum to Clark 63 soybeans formed small ineffective nodules which had very low nitrogenase activity compared to nodules formed by two effective strains, 110 Tet^R and 138 Kan^R. Mean numbers of cells per milligram of nodule tissue for plants up to 34 days old were 7.7×10⁶ for 8-0 Str^R, 4.1×10⁸ for 110 Tet^R and 7.6×10⁸ for 138 Kan^R. Cell counts per unit mass of nodule were independent of plant age for strains 110 Tet^R and 138 Kan^R, however, for strain 8-0 Str^R, 25 and 34 days old plants had fewer viable cells per nodule mass than 18 day old plants. When a mixture of two effective strains was used, the nodules of individual plants were predominantly caused by either 110 Tet^R or 138 Kan^R. In one experiment the predominance was random, but in another, strain 110 Tet^R clearly dominated. Strain 138 Kan^R was absent in some nodules on 18 day old plants, and in others, less than 10² cells per nodule were found. When strains 8-0 Str^R and 138 Kan^R were used as mixed inoculum, most of the nodules had strain 8-0 Str^R but strain 138 Kan^R was detected in many nodules and was generally evident in the largest nodules. Nitrogenase activity by many individual nodules was low except for nodules which had cells of 138 Kan^R. Nitrogenase activity by whole root systems of these plants was relatively high and similar to plants that had only nodules of strain 138 Kan^R. Similar relationships were observed for a mixed inoculum of 8-0 Str^R and 110 Tet^R. In general, mixed inoculations resulted in nodules with a particular strain being dominant for each individual plant. Double infections within individual nodules were not uncommon and such nodules often had disproportionate numbers of cells of two competingR. japonicum strains.Contribution from the Laboratory of Soil Microbiology, Department of Agronomy, Missouri Agricultural Experiment Station. Missouri Journal Series Number 7967.     

Studies of conformational isomerism in α-melanocyte stimulating hormone by design of cyclic analogues

Results of energy calculations for α-MSH (α-melanocyte stimulating hormone, Ac-Ser¹-Tyr²-Ser³-Met⁴-Glu⁵-His⁶-Phe⁷-Arg⁸-Trp⁹-Gly¹⁰-Lys¹¹-Pro¹²-Val¹³-NH₂) and [D -Phe⁷]α-MSH were used for design of cyclic peptides with the general aim to stabilize different conformational isomers of the parent compound. The minimal structural modifications of the conformationally flexible Gly¹⁰ residue, as substitutions for L -Ala, D -Ala, or Aib (replacing of hydrogen atoms by methyl groups), were applied to obtain octa- and heptapeptide analogues of α-MSH(4–11) and α-MSH(5–11), which were cyclized by lactam bridges between the side chains in positions 5 and 11. Some of these analogues, namely those with substitutions of the Gly¹⁰ residue with L -Ala or Aib, showed biological activity potencies on frog skin comparable to the potency of the parent tridecapeptide hormone. Additional energy calculations for designed cyclic analogues were used for further refinement of the model for the biologically active conformations of the His-Phe-Arg-Trp “message” sequence within the sequences of α-MSH and [D -Phe⁷]α-MSH. In such conformations the aromatic moieties of the side chains of the His⁶, L/D -Phe⁷, and Trp⁹ residues form a continuous hydrophobic “surface,” presumably interacting with a complementary receptor site. This feature is characteristic for low-energy conformers of active cyclic analogues, but it is absent in the case of inactive analogues. This particular spatial arrangement of functional groups involved in the message sequence is very close for α-MSH and [D -Phe⁷]α-MSH, as well as for biologically active cyclic analogues despite differences of dihedral angle values for corresponding low-energy conformations. © 1998 John Wiley & Sons, Inc. Biopoly 46: 155–167, 1998     

A chalcone-based fluorescent chemosensor for detecting Mg2+ and Cd2+

SBOD (sodium (E)-2-(3-[5-bromothiophen-2-yl]-3-oxoprop-1-en-1-yl)-4,6-dichlorophenolate) was designed and synthesized as a chalcone-based fluorescent turn-on chemosensor for Mg²⁺ and Cd²⁺. SBOD selectively detected Mg²⁺ and Cd²⁺ through the increase in effective fluorescence. Detection limits of SBOD for Mg²⁺ and Cd²⁺ were calculated to be 3.8 μM and 2.9 μM, respectively. The binding modes of SBOD for Mg²⁺ and Cd²⁺ were determined to be 1:1 by ESI-MS and Job plot. Association mechanisms for SBOD to Mg²⁺ and Cd²⁺ were illustrated by ESI-MS, UV–vis, fluorescence spectroscopy, and calculations.     

The effects of transmitters on the affinity of the insoluble fraction of Ox brain for Na+ and K+

The insoluble fraction of ox-brain, which had previously been shown to have a non-linear affinity for Na⁺ and K⁺, was prepared. Acetylcholine (1×10^–8 mol/l and 1×10^–7 mol/l) reduced the affinity of the fraction for Na⁺ and K⁺ to zero, while at 1×10^–6 mol/l, the affinity for the cations was almost as high as in the absence of the transmitter; the affinities for Na⁺ and K⁺ were particularly high, when the supernatant concentrations of these ions exceeded 80–100 mM. Addition of eserine (3×10^–5 mol/l) considerably modified the response of the fraction to acetylcholine (1×10^–5 mol/l). Atropine (1×10^–8 mol/l) in the absence or presence of acetylcholine (1×10^–5, or 1×10^–4 mol/l) reduced the affinity of the fraction for Na⁺ and K⁺ to zero. Epinephrine (3×10^–10 mol/l) lowered the affinity for Na⁺ and K⁺, while ergotamine itself (1×10^–5 mol/l) reduced it to zero. The addition of both epinephrine and ergotamine at the latter concentrations restored the affinity of the fractions for Na⁺ and K⁺ to what it had been in the absence of the transmitter or antagonist, previously reported. Norepinephrine (3×10^–10 mol/l), or ouabain (1×10^–7 mol/l) reduced the affinity of the fraction for Na⁺ and K⁺ to zero. Thus, the transmitters and antagonists altered the affinity of the insoluble fraction for Na⁺ and K⁺ nonlinearity, dependent upon their concentrations, the concentrations of the cations, and the interaction of transmitter and antagonist.     

Purification and biochemical characterization of two extracellular peroxidases from Phanerochaete chrysosporium responsible for lignin biodegradation

Two extracellular peroxidases from Phanerochaete chrysosporium, namely a lignin peroxidase (LiP) and manganese peroxidase (MnP), were purified simultaneously by applying successively, ultrafiltration, ion-exchange and gel filtration chromatography. LiP and MnP have a molecular mass of 36 and 45 kDa, respectively. The optimal pHs for LiP and MnP activities were 3.0 and 4.5, respectively. Both peroxidases showed maximal activity at 30 °C and moderate thermostability. MnP activity was strongly inhibited by Fe²⁺, Zn²⁺, Mg²⁺ and Hg²⁺, and enhanced by Mn²⁺, Ca²⁺ and Cu²⁺. LiP activity was enhanced by Ca²⁺, Na⁺ and Co²⁺ and it was inhibited in the presence of K⁺, Hg⁺, Fe²⁺, Mg²⁺ and high concentrations of Cu²⁺ and Zn²⁺. The K_m and V_max for LiP toward veratryl alcohol as a substrate were 0.10 mM and 15.2 U mg⁻¹, respectively and for MnP toward Mn²⁺, they were respectively 0.03 mM and 25.5 U mg⁻¹. The two peroxidases were also able to break down rice lignin in a small-scale solid state treatment system. Data suggest these two peroxidases may be considered as potential candidates for the development of enzyme-based technologies for lignin degradation.     

红花草莓的组织培养与快繁技术研究

以红花草莓叶片为外植体,通过筛选诱导愈伤组织、不定芽及壮苗、生根的培养基,建立一套实用且易推广的红花草莓组培快繁技术体系。结果表明:在愈伤组织的诱导过程中TDZ的诱导效果优于6-BA,TDZ与NAA配合使用效果优于与IBA的组合。6-BA浓度为0.5 mg·L~(-1)时不定芽诱导率高达86.6%。低浓度的6-BA和8 g·L~(-1)的琼脂更有利于壮苗培养,NAA比IBA更有利诱导生根。综上述,最适红花草莓愈伤组织的诱导培养基为MS+1.0 mg·L~(-1)TDZ+0.5 mg·L~(-1)NAA+30 g·L~(-1)蔗糖+7 g·L~(-1)琼脂;最适不定芽分化的培养基为MS+0.5 mg·L~(-1)6-BA+0.1 mg·L~(-1)NAA+30 g·L~(-1)蔗糖+7 g·L~(-1)琼脂;最适壮苗培养基为MS+0.1 mg·L~(-1)6-BA+0.1 mg·L~(-1)NAA+30 g·L~(-1)蔗糖+8g·L~(-1)琼脂;最适生根培养基为MS+0.5mg·L~(-1)NAA+30 g·L~(-1)蔗糖+8 g·L~(-1)琼脂。试管苗移栽生长20 d后,成活率高达93%,且后期草莓苗生长壮健。此体系的建立为优质红花草莓种苗大规模生产提供了科学依据和技术支持。     

Effects of NaCl on ion relations and carbohydrate status of roots and on osmotic regulation of roots and shoots of Atriplex amnicola

Abstract Atriplex amnicola was grown at 25, 200 or 400 mol m³ NaCl. Root tissues at different stages of development were investigated for concentrations of K⁺, Na⁺ and Mg²⁺, and in some cases for Cl^?. Sugar and starch concentrations were measured for plants grown at 25 or 400 mol m³ NaCl. In the ‘slightly vaeuolated’ root tips, Na⁺ was only 40 mol m^?3 at an external concentration of 400 mol m^?3 NaCl. The concentrations of K⁺ were not affected substantially by external NaCl between 25 mol m^?3 and 400 mol m^?3. The ‘highly vacuolated’ root tissues had substantially higher concentrations of K⁺, Na⁺ and Cl^? in plants grown at 200 and 400 mol m ³ NaCl than in plants grown at 25 mol m^?3 NaCl. Concentrations of Cr and of the sum of the cations in recently expanded tissue were similar to those in the bulk of the roots, consisting mainly of old cells. However, the K⁺: Na⁺ decreased with age; at 400 mol m^?3 external NaCl with a K⁺: Na⁺ of 0.012, the K⁺: Na⁺ in recently expanded 12 mm root tips was as high as 1.6, compared with 0.7 for the bulk of the roots. These ion data were used to estimate cytoplasmic and vacuolar concentrations of K⁺ and Na ⁺. Such calculations indicated that between 25 mol m³ and 400 mol m^?3 external NaCl the concentration of the sum of (Na⁺+K⁺) in the cytoplasm was maintained at about 180–200 mol m^?3 (cell water basis). In contrast, the (Na⁺+ K⁺) concentration in the vacuole was 170 mol m^?3 for plants grown at 25 mol m^?3 NaCl and 420 mol 400 mol m^?3 NaCl. The expanding root (issues exhibited greatly decreased soluble sugars and starch between dusk and dawn. Ai both times, sugar and starch concentrations in these tissues were 2.5–4.0 times greater in plants grown at 400 mol m^?3 NaCl compared with plants grown at 25 mol m^?3 NaCl. In contrast, carbohydrate concentrations in expanded root tissues were very similar at 25 and 400 mol m^?3 and showed little diurnal fluctuation. This paper considers the causes for the slower growth of A. amnicola at 400 than at 25 mol m”³ NaCl, using the data for the roots described here, and those for the shoots presented in the preceding paper (Aslam et al., 1986). There is no support for possible adverse effects by high internal ion concentrations. Instead, there may be deficiencies in supply of organic solutes for osmotic regulation; during part of the night a limited supply of such solutes may well restrict the rate of expansion of cells in plants growing at 400 mol m^?3 NaCl. There is insufficient evidence to decide whether this limitation in the expanding tissues is particularly prominent for the roots or for the shoots.