An isotopic method for testing the influence of leaf litter quality on carbon fluxes during decomposition

During microbial breakdown of leaf litter a fraction of the C lost by the litter is not released to the atmosphere as CO₂ but remains in the soil as microbial byproducts. The amount of this fraction and the factors influencing its size are not yet clearly known. We performed a laboratory experiment to quantify the flow of C from decaying litter into the soil, by means of stable C isotopes, and tested its dependence on litter chemical properties. Three sets of ¹³C-depleted leaf litter (Liquidambar styraciflua L., Cercis canadensis L. and Pinus taeda L.) were incubated in the laboratory in jars containing ¹³C-enriched soil (i.e. formed C4 vegetation). Four jars containing soil only were used as a control. Litter chemical properties were measured using thermogravimetry (Tg) and pyrolysis–gas chromatography/mass spectrometry–combustion interface–isotope ratio mass spectrometry (Py–GC/MS–C–IRMS). The respiration rates and the δ¹³C of the respired CO₂ were measured at regular intervals. After 8 months of incubation, soils incubated with both L. styraciflua and C. canadensis showed a significant change in δ¹³C (δ¹³C_final = −20.2 ± 0.4‰ and −19.5 ± 0.5‰, respectively) with respect to the initial value (δ¹³C_initial = −17.7 ± 0.3‰); the same did not hold for soil incubated with P. taeda (δ¹³C_final:−18.1 ± 0.5‰). The percentages of litter-derived C in soil over the total C loss were not statistically different from one litter species to another. This suggests that there is no dependence of the percentage of C input into the soil (over the total C loss) on litter quality and that the fractional loss of leaf litter C is dependent only on the microbial assimilation efficiency. The percentage of litter-derived C in soil was estimated to be 13 ± 3% of total C loss.     

Complex and Tandem Repeat Structure of Subtelomeric Regions in the Taiwan Cricket, <Emphasis Type="BoldItalic">Teleogryllus taiwanemma</Emphasis>

Telomeres of most insects are composed of simple (TTAGG) _n repeats that are synthesized by telomerase. However, in some dipteran insects such as Drosophila melanogaster, (TTAGG) _n repeats or telomerase activity has not been detected. Although telomere structure is well documented in Diptera and Lepidoptera, very limited information is available on lower insect groups. To understand general aspects of telomere function and evolution in insects, we endeavored to characterize structures of the telomeric and subtelomeric regions in a lower insect, the Taiwan cricket, Teleogryllus taiwanemma. FISH analysis of this insect's chromosomes demonstrated (TTAGG) _n repeat elements in all distal ends. Just proximal to the telomeric repeats, the highly conserved 9-kb long terminal unit (LTU) sequences are tandemly repeated. These were observed in four of six chromosomes, three autosomal ends, and one X-chromosomal end. LTU sequences represent about 0.2% of the T. taiwanemma genome. Each LTU contains a core (TTAGG)₈-like sequence (TRLS) and five types of conserved sequences—ST (short telomere associated), J (joint), X, SR (satellite sequence rich), and Y—which vary in length from about 150 bp to 2.7 kb. The LTU sequence is defined as ST–J–TRLS–SR–X–Y–X–Y–X. Most LTU regions may be derived from the ancestral common sequence, which is observed in ST regions six times and at many other LTU sites. We could not find the LTU-like sequence in three other crickets including the closest species, T. emma, suggesting that the LTU in T. taiwanemma has been rapidly amplified in subtelomeric regions through recent evolutional events. It is also suggested that the highly conserved structure of the LTU is maintained by recombination and may contribute to telomere elongation, as seen in dipteran insects. Received: 6 August 2001/Accepted: 10 October 2001     

Somatic embryogenic tissue establishment from mature Pinus nigra Arn. ssp. salzmannii embryos

Summary Somatic embryogenesis from mature zygotic embryos of salgare?o pine (Pinus nigra Arn. ssp. salzmannii) was induced after 2 wk of culture in L₁ medium [Murashige and Skoog mineral solution with 10.7 μM naphthaleneacetic acid (NAA) and 8.8 μM 6-benzyladenine (BA)] or L₂ medium [Gupta and Durzan mineral solution (DCR)] supplemented with the phytohormone combination as above. Four different combinations of growth regulators, NAA (2.6–10.7 μM) and BA (6.6–8.8 μM), were tried for subsequent passage. The best percentage of embryo induction and manifestation was obtained on DCR medium with 2.6 μM NAA and 6.6–8.8 μM BA. Transfer of isolated somatic embryos from the basal media to L₃ medium (Quoirin and Le Poivre modified mineral solution without hormones and supplemented with 0.3% activated charcoal), facilitated random embryo maturation and some development into plantlets.     

Changes in trophic level of Squatina guggenheim with increasing body length: relationships with type, size and trophic level of its prey

The occurrence of changes in the trophic level (TL) of sharks with growth has not been quantified until now. Here length-related changes on Squatina guggenheim Marini trophic level were determined, and shifts in type, size and trophic level of its prey were analysed. Sampling took place during five bottom trawl surveys conducted in the Argentine–Uruguayan Common Fishing Zone during spring (December/1995, October/1997) and fall (March/1997, March–April/1998, May–June/1998), using an Engel bottom-trawl net to capture the sharks. Three length groups were defined based on diet composition and using a cluster analysis (group I, 23–60 cm; group II, 61–80 cm; group III, 81–91 cm L _T). An ANOSIM procedure detected significant differences (P < 0.05) in the diet spectrum between the three length groups. The smallest sharks (group I) ingested fish prey ranging from 5 to 21 cm L _T, medium sharks (group II) fed on fish prey between 11 and 35 cm L _T, and largest sharks (group III) preyed on fish between 13 and 40 cm L _T. Diet structure of length groups were discriminated by almost the same prey taxa that characterized them. The increase of S. guggenheim body length promoted a decrease in the relative importance of small pelagic fishes. Contrarily, prey as medium benthopelagic fishes, medium pelagic squid and medium benthopelagic fishes showed an inverse tendency, indicating a broad diet spectrum of adults. Predator-length and prey-length relationship indicated a trend where 44.8% of S. guggenheim diet was integrated by prey <20% of their own body length and 32.8% of their diet was composed by prey >30% of their own length. The increase of mean prey weight was associated with the increase of predator weight and length. Smallest sharks (group I) were identified as secondary consumers (TL < 4) whereas medium sharks (group II) and largest sharks (group III) were placed as tertiary consumers (TL > 4). The study revealed an increase in S. guggenheim TL with shark growth as a consequence of changes on type, size and TL of prey ingested.     

Comparative Tolerances of Various <Emphasis Type="Italic">Beauveria bassiana</Emphasis> Isolates to UV-B Irradiation with a Description of a Modeling Method to Assess Lethal Dose

The tolerances of 20 Beauveria bassiana isolates derived from host insects worldwide to UV-B irradiation were assessed quantitatively in multi-dose bioassays. Conidial suspensions of the isolates smeared on glass slides were exposed to the gradient UV-B doses of 0.1–1.6 J cm⁻² (D), which generated from 0.75 to 10.17 min irradiation of weighted 312-nm wavelength at 2.0–2.61 mW cm⁻². Irradiated conidia were then incubated for 24 h at 25°C under saturated humidity. The ratio of germination at each dose over that in the blank control was defined as survival index (I _s). For all isolates, the I _s − D observations fit well with the survival model I _s = 1/[1 + exp(a + bD)] (0.94 ≤ r ² ≤ 0.99) generated widely spanned lethal doses of 0.154–0.928, 0.240–1.139, and 0.383–1.493 J cm⁻² for their losses of 50%, 75%, and 95% viabilities, respectively. These were far below the solar UV-B dose of 2.439 J cm⁻² measured in a sunny day during the summer. The large variation of UV-B tolerance among the isolates indicates a necessity to select UV-tolerant candidates for formulations applied to insect control during summer. The highly efficient bioassay method was developed to measure accurately the UV-B tolerances of fungal biocontrol agents as lethal doses.     

Functional characterisation of an engineered multidomain human P450 2E1 by molecular Lego

The human cytochrome P450s constitute an important family of monooxygenase enzymes that carry out essential roles in the metabolism of endogenous compounds and foreign chemicals. We present here results of a fusion between a human P450 enzyme and a bacterial reductase that for the first time is shown does not require the addition of lipids or detergents to achieve wild-type-like activities. The fusion enzyme, P450 2E1–BMR, contains the N-terminally modified residues 22–493 of the human P450 2E1 fused at the C-terminus to residues 473–1049 of the P450 BM3 reductase (BMR). The P450 2E1–BMR enzyme is active, self-sufficient and presents the typical marker activities of the native human P450 2E1: the hydroxylation of p-nitrophenol (K _M=1.84±0.09 mM and k _cat of 2.98±0.04 nmol of p-nitrocatechol formed per minute per nanomole of P450) and chlorzoxazone (K _M=0.65±0.08 mM and k _cat of 0.95±0.10 nmol of 6-hydroxychlorzoxazone formed per minute per nanomole of P450). A 3D model of human P450 2E1 was generated to rationalise the functional data and to allow an analysis of the surface potentials. The distribution of charges on the model of P450 2E1 compared with that of the FMN domain of BMR provides the ground for the understanding of the interaction between the fused domains. The results point the way to successfully engineer a variety of catalytically self-sufficient human P450 enzymes for drug metabolism studies in solution.     

Artificially increased ascorbate content affects zeaxanthin formation but not thermal energy dissipation or degradation of antioxidants during cold-induced photooxidative stress in maize leaves

Infiltrating detached maize (Zeamays L.) leaves with L-galactono-1,4-lactone (L-GAL) resulted in a 4-fold increase in the content of leaf ascorbate. Upon exposure to high irradiance (1000 μmol photons m⁻² s⁻¹) at 5 °C, L-GAL leaves de-epoxidized the xanthophyll-cycle pigments faster than the control leaves; the maximal ratio of de-epoxidized xanthophyll-cycle pigments to the whole xanthophyll-cycle pool was the same in both leaf types. The elevated ascorbate content, together with the faster violaxanthin de-epoxidation, did not affect the degree of photoinhibition and the kinetics of the recovery from photoinhibition, assayed by monitoring the maximum quantum efficiency of photosystem II primary photochemistry (F_v/F_m). Under the experimental conditions, the thermal energy dissipation seems to be zeaxanthin-independent since, in contrast to the de-epoxidation, the decrease in the efficiency of excitation-energy capture by open photosystem II reaction centers (F_v′/F_m′) during the high-irradiance treatment at low temperature showed the same kinetic in both leaf types. This was also observed for the recovery of the maximal fluorescence after stress. Furthermore, the elevated ascorbate content did not diminish the degradation of pigments or α-tocopherol when leaves were exposed for up to 24 h to high irradiance at low temperature. Moreover, a higher content of ascorbate appeared to increase the requirement for reduced glutathione. Received: 20 May 1999 / Accepted: 29 October 1999     

Optimization of Hydrogels for Transdermal Delivery of Lisinopril by Box–Behnken Statistical Design

The aim of this study was to investigate the combined influence of three independent variables on the permeation kinetics of lisinopril from hydrogels for transdermal delivery. A three-factor, three-level Box–Behnken design was used to optimize the independent variables, Carbopol 971 P (X ₁), menthol (X ₂), and propylene glycol (X ₃). Fifteen batches were prepared and evaluated for responses as dependent variables. The dependent variables selected were cumulative amount permeated across rat abdominal skin in 24 h (Q ₂₄; Y ₁), flux (Y ₂), and lag time (Y ₃). Aloe juice has been first time investigated as vehicle for hydrogel preparation. The ex vivo permeation study was conducted using Franz diffusion cells. Mathematical equations and response surface plots were used to relate the dependent and independent variables. The regression equation generated for the cumulative permeation of LSP in 24 h (Q ₂₄) was Y ₁ = 1,443.3–602.59X ₁ + 93.24X ₂ + 91.75X ₃ − 18.95X ₁ X ₂ – 140.93X ₁ X ₃ – 4.43X ₂ X ₃ – 152.63X ₁ ² – 150.03X₂ ² − 213.9X ₃ ². The statistical validity of the polynomials was established, and optimized formulation factors were selected by feasibility and grid search. Validation of the optimization study with 15 confirmatory runs indicated high degree of prognostic ability of response surface methodology. The use of Box–Behnken design approach helped in identifying the critical formulation parameters in the transdermal delivery of lisinopril from hydrogels.     

12-Oxophytodienoate reductase 3 (OPR3) is the isoenzyme involved in jasmonate biosynthesis

 In addition to OPR1 and OPR2, two isoenzymes of 12-oxophytodienoate reductase, a third isoform (OPR3) has recently been identified in Arabidopsis thaliana (L.) Heynh. The expression of the OPR3 gene is induced not only by a variety of stimuli, such as touch, wind, wounding, UV-light and application of detergent, but also by brassinosteroids. The three enzymes were expressed in a functional form in Escherichia coli, and OPR2 was additionally expressed in insect cell cultures and overexpressed in A. thaliana. Substrate conversion was analyzed using a stereospecific assay. The results show that OPR3 effectively converts the natural (9S,13S)-12-oxophytodienoic acid [K _m = 35 μM, V _max 53.7 nkat (mg protein)⁻¹] to the corresponding 3-2(2′(Z)-pentenyl) cyclopentane-1-octanoic acid (OPC-8:0) stereoisomer while OPR1 and OPR2 convert (9S,13S)-12-oxophytodienoic acid with greatly reduced efficiency compared to OPR3. Thus, OPR3 is the isoenzyme relevant for jasmonate biosynthesis. Received: 21 October 1999 / Accepted: 10 December 1999     

Inoculation and nitrate alter phytohormone levels in soybean roots: differences between a supernodulating mutant and the wild type

 The levels of different cytokinins, indole-3-acetic acid (IAA) and abscisic acid (ABA) in roots of Glycine max [L.] Merr. cv. Bragg and its supernodulating mutant nts382 were compared for the first time. Forty-eight hours after inoculation with Bradyrhizobium, quantitative and qualitative differences were found in the root's endogenous hormone status between cultivar Bragg and the mutant nts382. The six quantified cytokinins, ranking similarly in each genotype, were present at higher concentrations (30–196% on average for isopentenyl adenosine and dihydrozeatin riboside, respectively) in mutant roots. By contrast, the ABA content was 2-fold higher in Bragg, while the basal levels of IAA [0.53 μmol (g DW)⁻¹, on average] were similar in both genotypes. In 1 mM NO₃ ⁻-fed Bragg roots 48 h post-inoculation, IAA, ABA and the cytokinins isopentenyl adenine, and isopentenyl adenosine quantitatively increased with respect to uninoculated controls. However, only the two cytokinins increased in the mutant. High NO₃ ⁻ (8 mM) markedly reduced root auxin concentration, and neither genotypic differences nor the inoculation-induced increase in auxin concentration in Bragg was observed under these conditions. Cytokinins and ABA, on the other hand, were little affected by 8 mM NO₃ ⁻. Root IAA/cytokinin and ABA/cytokinin ratios were always higher in Bragg relative to the mutant, and responded to inoculation (mainly in Bragg) and nitrate (both genotypes). The overall results are consistent with the auxin-burst-control hypothesis for the explanation of autoregulation and supernodulation in soybean. However, they are still inconclusive with respect to the inhibitory effect of NO₃ ⁻. Received: 16 April 1999 / Accepted: 13 December 1999     

The stability of the molybdenum-azotochelin complex and its effect on siderophore production in Azotobacter vinelandii

 Azotobacter vinelandii produces five siderophores with different metal binding properties, depending on the concentrations of Fe(III) and molybdate in the growth medium. The three lower protonation constants of the unusual bis(catecholamide) siderophore azotochelin (L) were determined by a simultaneous spectrophotometric and potentiometric titration as log K ₅=3.65(5), log K ₄=7.41(3) and log K ₃=8.54(4). The metal-ligand equilibrium constant for [MoO₂(L)]^3– was obtained from analysis of the absorbance concentration data: at 20  °C and pH 6.6, log K _eq=4(1). Based on an average log K _a value of 12.1 for the two basic phenolic oxygens of azotochelin, the equilibrium formation constant was converted into the conventional formation constant K _f(MoL) = [MoO₂L^{3 –}]/[MoO₂ ²⁺][L^{5 –}] = 10³⁵ M^–1. To assess the influence of molybdenum-siderophore interactions on metal uptake in A. vinelandii, the dose-response effect of molybdate in the growth medium on siderophore biosynthesis was followed by UV-vis spectroscopy and HPLC. It could be shown that the formation of molybdenum siderophore complexes clearly reduces the concentration of free siderophores available for iron solubilization. Furthermore, in media with initial molybdate concentrations up to 100 μM, the molybdenum azotochelin complex is the predominant molybdenum species, suggesting that azotochelin might also possess sequestering activity towards molybdenum. Even higher molybdate levels result in a complete repression of the synthesis of the tetradentate siderophore azotochelin, while they initiate the alternative release of the more efficient iron chelator, the hexadentate siderophore protochelin. Received: 20 April 1998 / Accepted: 29 June 1998     

Type I blue copper proteins as enantioselective quenchers of the photoluminescence of Δ,Λ-Eu(pyridine-2,6-dicarboxylate)3 3–: azurin from Pseudomonas aeruginosa and its Met44→Lys mutant, amicyanin from Paracoccus versutus and parsley plastocyanin

 The dynamic quenching of the luminescence of racemic Eu(III)(pyridine-2,6-dicarboxylate=dpa)₃ ^3– by the title proteins is investigated and the enantioselectivity of the proteins in the quenching of the Δ and Λ enantiomers of Eu(dpa)₃ ^3– is determined. The two diastereomeric quenching rate constants pertaining to azurin (k _q ^Δ=3.3×10⁶, k _q ^Λ=2.7×10⁶ M^–1s^–1, pH 7.2, ionic strength I=22 mM) are lower than for its Met→44Lys mutant (k _q ^Δ=1.9×10⁷, k _q ^Λ=1.4×10⁷ M^–1 s^–1, same pH and I), indicating that energy transfer occurs from Eu(dpa)₃ ^3– to the Cu(II) centre when the luminophore is bound to the hydrophobic patch of the protein near residue 44. The enantioselectivity remains unaltered by the mutation: k _q ^Δ/k _q ^Λ=1.27±0.04, so Lys44 is probably not in direct contact with the Eu chelate. The I and pH dependence of k _q indicate that the lysine residue interacts electrostatically with Eu(dpa)₃ ^3–. For plastocyanin the quenching rates are of the order of 10⁶ M^–1 s^–1; for amicyanin they are two orders of magnitude larger (k _q ^Δ=12×10⁷, k _q ^Λ=11×10⁷ M^–1 s^–1, pH 7.2, I=22 mM). The variation of k _q is attributed to differences in the charge distribution on the proteins, which influences the binding of the luminophore to the protein surface. For amicyanin the anion binding site near Lys59 and Lys60 may be involved in the energy transfer. Received: 16 June 1998 / Accepted: 18 September 1998     

Intraprotoplasmic and wall-localised formation of arabinoxylan-bound diferulates and larger ferulate coupling-products in maize cell-suspension cultures

Maize (Zea mays L.) cell cultures incorporated radioactivity from [¹⁴C]cinnamate into hydroxycinnamoyl-CoA derivatives and then into polysaccharide-bound feruloyl residues. Within 5–20 min, the CoA pool had lost its ¹⁴C by turnover and little or no further incorporation into polysaccharides then occurred. The system was thus effectively a pulse–chase experiment. Kinetics of radiolabelling of diferulates (also known as dehydrodiferulates) varied with culture age. In young (1–3 d) cultures, polysaccharide-bound [¹⁴C]feruloyl- and [¹⁴C]diferuloyl residues were both detectable within 1 min of [¹⁴C]cinnamate feeding. Thus, feruloyl residues were dimerised <1 min after their attachment to polysaccharides. For at least the first 2.3 h after [¹⁴C]cinnamate feeding, polysaccharide-bound [¹⁴C]diferuloyl residues remained almost constant at ≈7% of the total polysaccharide-bound [¹⁴C]ferulate derivatives. Since feruloyl residues are attached to polysaccharides <1 min after the biosynthesis of the latter, and >10 min before secretion, the data show that extensive feruloyl coupling occurred intra-protoplasmically. Exogenous H₂O₂ (1 mM) caused little additional feruloyl coupling; therefore, wall-localised coupling may have been peroxidase-limited. In older (e.g. 4 d) cultures, less intraprotoplasmic coupling occurred: during the first 2.5 h, polysaccharide-bound [¹⁴C]diferuloyl residues were a steady 1.4% of the total polysaccharide-bound [¹⁴C]ferulate derivatives. In contrast to the situation in younger cultures, exogenous H₂O₂ induced a rapid 4- to 6-fold increase in all coupling products, indicating that coupling in the walls was H₂O₂-limited. In both 2- and 4-d-old cultures, polysaccharide-bound ¹⁴C-trimers and larger coupling products exceeded [¹⁴C]diferulates 3- to 4-fold, but followed similar kinetics. Thus, although all known dimers of ferulate can now be individually quantified, it appears to be trimers and larger products that make the major contribution to cross-linking of wall polysaccharides in cultured maize cells. We argue that feruloyl arabinoxylans that are cross-linked before and after secretion are likely to loosen and tighten the cell wall, respectively. The consequences for the control of cell expansion and for the response of cell walls to an oxidative burst are discussed. Received: 19 January 2000 / Accepted: 13 April 2000     

Abscisic acid and hydraulic conductivity of maize roots: a study using cell- and root-pressure probes

Using root- and cell-pressure probes, the effects of the stress hormone abscisic acid (ABA) on the water-transport properties of maize roots (Zea mays L.) were examined in order to work out dose and time responses for root hydraulic conductivity. Abscisic acid applied at concentrations of 100–1,000 nM increased the hydraulic conductivity of excised maize roots both at the organ (root Lp_r: factor of 3–4) and the root cell level (cell Lp: factor of 7–27). Effects on the root cortical cells were more pronounced than at the organ level. From the results it was concluded that ABA acts at the plasmalemma, presumably by an interaction with water channels. Abscisic acid therefore facilitated the cell-to-cell component of transport of water across the root cylinder. Effects on cell Lp were transient and highly specific for the undissociated (+)-cis-trans-ABA. The stress hormone ABA facilitates water uptake into roots as soils start drying, especially under non-transpiring conditions, when the apoplastic path of water transport is largely excluded. Received: 26 February 2000 / Accepted: 17 August 2000     

Patterns of embryonic neurogenesis in a primitive wingless insect, the silverfish, Ctenolepisma longicaudata: comparison with those seen in flying insects

 Neurogenesis was examined in the central nervous system of embryos of the primitively wingless insect, the silverfish, Ctenolepisma longicaudata, using staining with toluidine blue (TB) and the incorporation of bromodeoxyuridine (BUdR). The silverfish has the same number and positioning of neuroblasts as seen in more advanced insects and the relative order in which the different neuroblasts segregate from the neuroectoderm is highly conserved between Ctenolepisma and the grasshopper, Schistocerca. Of the 31 different neuroblasts found in a thoracic segment, one (NB 6–3) has a much longer proliferative period in silverfish. Of the remainder, 14 have similar proliferative phases, while16 neuroblasts have extended their proliferative period by 10% of embryogenesis or greater in the grasshopper as compared with the silverfish. Both insects had similar periods of abdominal neurogenesis except that in the silverfish terminal ganglion a prominent set of neuroblasts continued dividing until close to hatching, possibly reflecting the importance of cercal sensory input in this insect. This comparison between silverfish and grasshopper shows that the shift from wingless to flying insects was not accompanied by the addition of any new neuronal lineages in the thorax. Instead, selected lineages underwent a proliferative expansion to supply the additional neurons presumably needed for flight. The expansion of specific thoracic lineages was accompanied by the reduction of the terminal abdominal lineages as flying insects began to de-emphasize their cercal sensory system. Received:16 March 1998 / Accepted: 21 May 1998     

Effects of trans-pyridine ligands on the interactions of RuII and RuIII ammine complexes N7-coordinated to purine nucleosides and DNA

 DNA binding by trans-[(H₂O)(Pyr)(NH₃)₄Ru^II]²⁺ (Pyr=py, 3-phpy, 4-phpy, 3-bnpy, 4-bnpy) is highly selective for G⁷ with K _G=1.1×10⁴ to 2.8×10⁴, with the more hydrophobic Pyr ligands exhibiting slightly higher binding. A strong dependence on ionic strength indicates that ion-pairing with DNA occurs prior to binding. At μ=0.05, d[Ru^II-DNA]/dt=k[Ru^II][DNA], where k=0.17–0.21 M^–1s^–1 with the various Pyr ligands. The air oxidation of [(py)(NH₃)₄Ru^II] _n -DNA to [(py)(NH₃)₄Ru^III] _n -DNA at pH 6 occurs with a pseudo-first-order rate constant of k _obs=5.6×10^–4s^–1 at μ=0.1, T=25  °C. Strand cleavage of plasmid DNA appears to occur by both Fenton/Haber-Weiss chemistry and by base-catalyzed routes, some of which are independent of oxygen. Base-catalyzed cleavage is more efficient than O₂ activation at neutral pH and involves the disproportionation of covalently bound Ru^III and, in the presence of O₂, Ru-facilitated autoxidation to 8-oxoguanine. Disproportionation of [py(NH₃)₄Ru^III] _n -DNA occurs according to the rate law: d[Ru^II–G_DNA]/dt=k ₀[Ru^III–G_DNA]+k ₁[Ru^III–G_DNA][OH^–], where k ₀=5.4×10^–4s^–1 and k ₁=8.8 M^–1s^–1 at 25  °C, μ=0.1. The appearance of [(Gua)(py)(NH₃)₄Ru^III] under argon, which occurs according to the rate law: d[Ru^III–G]/dt=k ₀[Ru^III–G_DNA]+k ₁[OH^–][Ru^III–G_DNA] (k ₀=5.74×10^–5 s^–1, k ₁=1.93×10^–2M^–1s^–1 at T=25  °C, μ=0.1), is consistent with lysis of the N-glycosidic bond by Ru^IV-induced general acid hydrolysis. In air, the ratio of [Ru-8-OG]/[Ru-G] and their net rates of appearance are 1.7 at pH 11, 25  °C. Small amounts of phosphate glycolate indicate a minor oxidative pathway involving C4′ of the sugar. In air, a dynamic steady-state system arises in which reduction of Ru^IV produces additional Ru^II. Received: 11 November 1998 / Accepted: 3 March 1999     

Ferric reduction by iron-limited Chlamydomonas cells interacts with both photosynthesis and respiration

Iron limitation led to a large increase in extracellular ferricyanide (Fe[III]) reductase activity in cells of the green alga Chlamydomonas reinhardtii Dangeard. Mass-spectrometric measurement of gas exchange indicated that ferricyanide reduction in the dark resulted in a stimulation of respiratory CO₂ production without affecting the rate of respiratory O₂ consumption, consistent with the previously postulated activation of the oxidative pentose phosphate pathway in support of Fe(III) reduction by iron-limited Chlamydomonas cells (X. Xue et al., 1998, J. Phycol. 34: 939–944). At saturating irradiance, the rate of ferricyanide reduction was stimulated almost 3-fold, and this stimulation was inhibited by 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea. Ferricyanide reduction during photosynthesis resulted in approximately a 50% inhibition of photosynthetic CO₂ fixation at saturating irradiance, and almost 100% inhibition of CO₂ fixation at sub-saturating irradiance. Photosynthesis by iron-sufficient cells was not affected by ferricyanide addition. Addition of 250 μM ferricyanide to iron-limited cells in which photosynthesis was inhibited (either by the presence of glycolaldehyde, or by maintaining the cells at the CO₂ compensation point) resulted in a stimulation in the rate of gross photosynthetic O₂ evolution. Chlorophyll a fluorescence measurements indicated a large increase in non-photochemical quenching during ferricyanide reduction in the light; the increase in nonphotochemical quenching was abolished by the addition of nigericin. These results suggest that reduction of extracellular ferricyanide (mediated at the plasma membrane) interacts with both photosynthesis and respiration, and that both of these processes contribute NADPH in the light. Received: 15 September 1999 / Accepted: 14 October 1999     

Kinetics of formation and stability of {Pt(dien)}2+ complexes with octamer and 14-mer DNA oligonucleotides containing a GG sequence

 Reaction of [Pt(dien)Cl]⁺ (1) with the 14-mer oligonucleotide 5′-d(ATACATGGTACATA) (I) gave rise to two major species which corresponded to the 5′-G and 3′-G platinated monofunctional adducts, and a minor amount of the bis-platinated adduct formed during the later stages of the reaction. The reaction of (1) with the related octamer 5′-d(ATACATGG) (II) was also investigated. Kinetic data obtained by HPLC showed that the 5′-G and 3′-G bases of the 14-mer oligonucleotide were platinated at similar rates: the second-order rate constant is 53×10^–2 M^–1 s^–1 at 298 K in 0.1 M NaClO₄. However, the platination rate of 5′-G of the octamer (II) (k=69×10^–2 M^–1 s^–1) was enhanced by a factor of three compared to the rate of platination at 3′-G (k=22×10^–2 M^–1 s^–1). All the adducts were separated by HPLC and characterized by NMR spectroscopy, enzymatic digestion and MALDI-TOF mass spectrometry. ¹H and ¹⁵N NMR shifts suggest that there are distinct conformational differences between 14-mer duplexes platinated at 5′-G (I5′ _ds) and 3′–G (I3′ _ds). Molecular mechanics modelling indicates that rotation around the Pt-N7 bond is more restricted in the case of the 5′-G adduct than in that of the 3′-G adduct. The binding of {Pt(dien)}²⁺ to 5′-GN7 and 3′-GN7 in the monofunctional adducts of (I) was shown to be reversible upon the addition of high concentrations of chloride ions. Received: 3 July 1998 / Accepted: 10 November 1998     

Chlamydomonas contains calcium stores that are mobilized when phospholipase C is activated

Mastoparan induces Ca²⁺-dependent deflagellation of the unicellular green alga Chlamydomonas moewusii Gerloff, as well as the activation of phospholipase C and the production of inositol 1,4,5-trisphosphate (InsP₃; T. Munnik et al., 1998, Planta 207: 133–145). Even in the absence of extracellular Ca²⁺, mastoparan still induces deflagellation (L.M. Quarmby and H.C. Hartzell, 1994, J Cell Biol 124: 807–815; J.A.J. van Himbergen et al., 1999, J Exp Bot, in press) suggesting that InsP₃ mediates Ca²⁺ release from intracellular stores. To test this hypothesis, cells were pre-loaded with ⁴⁵Ca²⁺ and their plasma membranes permeabilized by digitonin. Subsequent treatment of the cells with mastoparan (3.5 μM) induced release of intracellular ⁴⁵Ca²⁺. Mastoparan also activated phospholipase C in permeabilized cells, as demonstrated by the breakdown of ³²P-phosphatidylinositol 4,5-bisphosphate and the production of diacylglycerol. The mastoparan analogues mas7 and mas17 were also effective and their efficacy was correlated with their biological activity. X-ray microanalysis showed that electron-dense bodies (EDBs) are a major Ca²⁺ store in  C. moewusii. Analysis of digitonin-permeabilized cells showed that EDBs lost calcium at digitonin concentrations that released radioactivity from ⁴⁵Ca²⁺-labelled cells, suggesting that ⁴⁵Ca²⁺ monitored the content of EDBs. X-ray microanaysis of living cells treated with mastoparan also revealed that calcium was released from EDBs. Received: 30 December 1998 / Accepted: 25 June 1999     

Influence of soil porosity on water use in<Emphasis Type="Italic"> Pinus taeda</Emphasis>

We analyzed the hydraulic constraints imposed on water uptake from soils of different porosities in loblolly pine (Pinus taeda L.) by comparing genetically related and even-aged plantations growing in loam versus sand soil. Water use was evaluated relative to the maximum transpiration rate (E _crit) allowed by the soil-leaf continuum. We expected that trees on both soils would approach E _crit during drought. Trees in sand, however, should face greater drought limitation because of steeply declining hydraulic conductivity in sand at high soil water potential (Ψ _S). Transport considerations suggest that trees in sand should have higher root to leaf area ratios (A _R:A _L), less negative leaf xylem pressure (Ψ _L), and be more vulnerable to xylem cavitation than trees in loam. The A _R:A _L was greater in sand versus loam (9.8 vs 1.7, respectively). This adjustment maintained about 86% of the water extraction potential for both soils. Trees in sand were more deeply rooted (>1.9 m) than in loam (95% of roots <0.2 m), allowing them to shift water uptake to deeper layers during drought and avoid hydraulic failure. Midday Ψ _L was constant for days of high evaporative demand, but was less negative in sand (–1.6 MPa) versus loam (–2.1 MPa). Xylem was more vulnerable to cavitation in sand versus loam trees. Roots in both soils were more vulnerable than stems, and experienced the greatest predicted loss of conductivity during drought. Trees on both soils approached E _crit during drought, but at much higher Ψ _S in sand (<–0.4 MPa) than in loam (<–1.0 MPa). Results suggest considerable phenotypic plasticity in water use traits for P. taeda which are adaptive to differences in soil porosity. Received: 28 December 1999 / Accepted: 31 March 2000