Inhibition of photosynthetic electron transfer by amphotericin B

The polyene antibiotic amphotericin B inhibits photosynthetic electron transfer by Class II maize mesophyll chloroplasts, from water to FeCN, DCIP and diquat but not to plastocyanin. Photosystem 1 activity is also inhibited by amphotericin B, but ferredoxin-NADP reductase activity is not affected. The activity of all the photosynthetic electron transfer systems inhibited by amphotericin B can be restored by the addition of carrier amounts of plastocyanin. The results suggest that amphotericin B inhibits photosynthetic electron transfer by acting only at the plastocyanin site in the chain, and that the primary site of reduction of FeCN and DCIP from water by Class II chloroplasts lies on the reducing side of photosystem 1.     

Inheritance of bipyridyl herbicide resistance in Arctotheca calendula and Hordeum leporinum

The mode of inheritance of resistance to bipyridyl herbicides in bipyridyl-resistant biotypes of Arctotheca calendula and of Hordeum leporinum was investigated. F₁ plants from reciprocal crosses between diquat-resistant and -susceptible plants of A. calendula showed an intermediate response to diquat application that was nuclearly inherited. Treatment of F₂ plants with 100 g ai ha^-1 of diquat or 800 g ai ha^-1 of paraquat killed all homozygous-susceptible plants, caused severe injury to heterozygous plants but only slight or no injury to homozygous-resistant plants. Back crosses of F₁ to susceptible plants exhibited intermediate and susceptible phenotypes. The observed segregation ratios in F₂ and test-cross populations fitted predicted segregation ratios, 1:2:1 (R:I:S) and 1:1 (I:S) respectively, showing that bipyridyl resistance is conferred by a single incompletely-dominant gene. Biotypes of paraquat-resistant and -susceptible H. leporinum were crossed reciprocally. F₁ plants from reciprocal crosses showed an intermediate response to paraquat application. The F₂ progeny showed segregation ratios that fitted the predicted segregation ratio of 1:2:1 (R:I:S) forinheritance of resistance being governed by a single partially-dominant gene.     

Anthroyl stearate as a fluorescent probe of chloroplast membranes

1. A reversible light-induced enhancement of the fluorescence of a “hydrophobic fluorophore”, 12-(9-anthroyl)-stearic acid (anthroyl stearate), is observed with chloroplasts supporting phenazine methosulfate, cyclic or 1,1′-ethylene-2,2′-dipyridylium dibromide (Diquat) pseudo-cyclic electron flow; no fluorescence change is observed when methyl viologen or ferricyanide are used as electron acceptors. The stearic acid moiety of anthroyl stearate is important for its localization and fluorescence response in the thylakoid membrane, since structural analogs of anthroyl stearate lacking this group do not show the same response.

2. This effect is decreased under phosphorylating conditions (presence of ADP, P_i, Mg²⁺), and completely inhibited by the uncoupler of phosphorylation NH₄Cl (5–10 mM), as well as the ionophores nigericin and gramicidin-D (both at 5 · 10⁻⁸ M). The MgCl₂ concentration dependence of the anthroyl stearate enhancement effect is identical to that previously observed for cyclic photophosphorylation, as well as for the formation of a “high energy intermediate”. The anthroyl stearate fluorescence enhancement is inhibited by increasing concentrations of ionophores in parallel with the decrease in ATP synthesis, but is essentially unaffected by specific inhibitors (Dio-9 and phlorizin) of photophosphorylation; thus, it appears that anthroyl stearate monitors a component of the “high energy state” of the thylakoid membrane rather than a terminal phosphorylation step.

3. The light-induced anthroyl stearate fluorescence enhancement is suggested to monitor a proton gradient in the energized chloroplast because (a) similar enhancement can be produced by sudden injection of hydrogen ions in a solution of anthroyl stearate; (b) when the proton gradient is dissipated by gramicidin or nigericin light-induced anthroyl stearate fluorescence is eliminated; (c) when the proton gradient is dissipated by tetraphenylboron, light-induced anthroyl stearate fluorescence decreases, and (d) light-induced anthroyl stearate fluorescence change as a function of pH is qualitatively similar to that observed with other probes for a proton gradient (e.g. 9-aminoacridine). Furthermore, anthroyl stearate does not monitor H⁺ uptake per se because (a) the pH dependence of H⁺ transport is different from that of the anthroyl stearate fluorescence change, and (b) tetraphenylboron, which does not inhibit H⁺ uptake, reduces anthroyl stearate fluorescence.

Thus, anthroyl stearate appears to be a useful probe of a proton gradient supported by phenazine methosulfate or Diquat catalyzed electron flow and is the first “non-amine” fluorescence probe utilized for this purpose in chloroplasts.     

Method for measurement of the quaternary amine compounds paraquat and diquat in human urine using high-performance liquid chromatography–tandem mass spectrometry

We have developed a highly selective and sensitive analytical method to quantify paraquat and diquat by use of high-performance liquid chromatography-tandem mass spectrometry (HPLC–MS/MS). The sample preparation includes solid phase extraction that uses weak cation exchange cartridges. These highly charged dual quaternary amines were not retained by standard reversed phase columns, but they could be adequately separated through HPLC with a HILIC column. The detection was carried out with a triple quadrupole mass spectrometer with an electrospray ionization probe in positive ion mode in multiple reaction monitoring. Repeated analysis in human urine samples spiked with low (5 ng/ml), medium (15 ng/ml), and high (30 ng/ml) concentrations of the analytes yielded relative standard deviations of less than 9%. The extraction efficiencies ranged from 77.7% to 94.2%. The limits of detection were in the range of 1 ng/ml.     

Nitric oxide counteracts cytotoxic processes mediated by reactive oxygen species in plant tissues

Many environmental conditions subject plants to oxidative stress, in which reactive oxygen species (ROS) are overproduced. These ROS act as transduction signals in plant defense responses, but also cause effects that result in cellular damage. Since nitric oxide (NO) is a bioactive molecule able to scavenge ROS, we analyzed its effect on some cytotoxic processes produced by ROS in potato (Solanum tuberosum L. cv. Pampeana) leaves. Two NO donors: (i) sodium nitroprusside and (ii) a mixed solution of ascorbic acid and NaNO₂, were able to prevent chlorophyll loss mediated by the methyl viologen herbicide diquat (a ROS generator), with effective concentrations falling between 10 and 100 μM of the donors. This protection was mimicked by thiourea and penicillamine, two antioxidant compounds. Residual products from NO generation and decomposition failed to prevent chlorophyll decline. A specific NO scavenger, the potassium salt of 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (carboxy-PTIO), arrested NO-mediated chlorophyll protection. In addition, some events mediated by ROS during infection of potato leaves with Phytophthora infestans (race 1, 4, 7, 8, 10, 11, mating type A2) were also examined. In this sense, NO proved to markedly decrease ion leakage and the number of lesions, indicative of cell death, produced upon infection in potato leaves. The NO-mediated decrease in ion leakage was also inhibited by carboxy-PTIO. Fragmentation of DNA diminished when P. infestans-infected potato leaves were treated with 100 μM SNP. These results suggest that, acting as an antioxidant, NO can strongly counteract many ROS-mediated cytotoxic processes in plants. Moreover, the evidence of NO functionality in the plant kingdom is strengthened by this work. Received: 18 December 1998 / Accepted: 19 January 1999     

The effect of temperature of the rate of photosynthetic electron transfer in chloroplasts of chilling-sensitive and chilling-resistant plants

1. Photochemical activities as a function of temperature have been compared in chloroplasts isolated from chilling-sensitive (below approximately 12 °C) and chilling-resistant plants.2. An Arrhenius plot of the photoreduction of NADP⁺ from water by chloroplasts isolated from tomato (Lycopersicon esculentum var. Gross Lisse), a chilling-sensitive plant, shows a change in slope at about 12 °C. Between 25 and 14 °C the activation energy for this reaction is 8.3 kcal·mole^?1. Between 11 and 3 °C the activation energy increases to 22 kcal·mole^?1. Photoreduction of NADP⁺ by chloroplasts from another chilling-sensitive plant, bean (Phaseolus vulgaris var. brown beauty), shows an increase in activation energy from 5.9 to 17.5 kcal·mole^?1 below about 12 °C.3. The photoreduction of NADP⁺ by chloroplasts isolated from two chilling-resistant plants, lettuce (Lactuca sativa var. winter lake) and pea (Pisum sativum var. greenfeast), shows constant activation energies of 5.4 and 8.0 kcal·mole^?1, respectively, over the temperature range 3–25 °C.4. The effect of temperature on photosynthetic electron transfer in the chloroplasts of chilling-sensitive plants is localized in Photosystem I region of photosynthesis. Both the photoreduction of NADP⁺ from reduced 2,6-dichlorophenol-indophenol and the ferredoxin-NADP⁺ reductase (EC 1.6.99.4) activity of choroplasts of chilling-sensitive plants show increases in activation energies at approximately 12 °C whereas Photosystem II activity of chloroplasts of chilling-sensitive plants shows a constant activation energy over the temperature range 3–25 °C. The photoreduction of Diquat (1,1′-ethylene-2,2′-dipyridylium dibromide) from water by bean chloroplasts, however, does not show a change in activation energy over the same temperature range. The activation energies of each of these reactions in chilling-resistant plants is constant between 3 and 25 °C.5. The effect of temperature on the activation energy of these reactions in chloroplasts from chilling-sensitive plants is reversible.6. In chilling-sensitive plants, the increased activation energies below approximately 12 °C, with consequent decreased rates of reaction for the photoreduction of NADP⁺, would result in impaired photosynthetic activity at chilling temperatures. This could explain the changes in chloroplast structure and function when chilling-sensitive plants are exposed to chilling temperatures.