Donato Pastore

Specific helium-neon laser sensitivity of the purified cytochrome c oxidase.

Purpose : In order to gain some insight into the mechanism of interaction between Helium-Neon (He-Ne) laser light and mitochondrial cytochromes, the sensitivity of cytochrome electron transfer activity to He-Ne laser was tested. Materials and methods : Irradiation of solutions containing either purified cytochromes or dissolved rat liver mitochondria was carried out (wavelength 632.8nm, fluence rate 10 m W cm -2, fluence 2 J cm -2) ; the irradiation conditions were the ones able to affect cytochrome c oxidase (COX) activity in mitochondria (Pastore et al., 1994). Results : Cytochrome c oxidation catalysed by COX was affected by He-Ne laser irradiation of the purified enzyme. This result was obtained from measurements of the pseudo-first-order kinetic constant and from determinations of the turnover number of the enzyme, performed at different cytochrome c/COX ratios. Consistently, the kinetic parameters of COX changed. On the contrary, no alteration in the rate of electron transfer catalysed by either cytochrome c or bc 1 complex was found. Conclusions : This study shows that purified COX is a specific target of He-Ne laser light; therefore, COX may be considered to be a mitochondrial photo-acceptor.PURPOSE In order to gain some insight into the mechanism of interaction between Helium-Neon (He-Ne) laser light and mitochondrial cytochromes, the sensitivity of cytochrome electron transfer activity to He-Ne laser was tested. MATERIALS AND METHODS Irradiation of solutions containing either purified cytochromes or dissolved rat liver mitochondria was carried out (wavelength 632.8 nm, fluence rate 10 mW cm(-2), fluence 2 J cm(-2)); the irradiation conditions were the ones able to affect cytochrome c oxidase (COX) activity in mitochondria (Pastore et al., 1994). RESULTS Cytochrome c oxidation catalysed by COX was affected by He-Ne laser irradiation of the purified enzyme. This result was obtained from measurements of the pseudo-first-order kinetic constant and from determinations of the turnover number of the enzyme, performed at different cytochrome c/COX ratios. Consistently, the kinetic parameters of COX changed. On the contrary, no alteration in the rate of electron transfer catalysed by either cytochrome c or bc1 complex was found. CONCLUSIONS This study shows that purified COX is a specific target of He-Ne laser light; therefore, COX may be considered to be a mitochondrial photo-acceptor.

The Existence of the K+ Channel in Plant Mitochondria

In this study, evidence is given that a number of isolated coupled plant mitochondria (from durum wheat, bread wheat, spelt, rye, barley, potato, and spinach) can take up externally added K+ ions. This was observed by following mitochondrial swelling in isotonic KCl solutions and was confirmed by a novel method in which the membrane potential decrease due to externally added K+ is measured fluorimetrically by using safranine. A detailed investigation of K+ uptake by durum wheat mitochondria shows hyperbolic dependence on the ion concentration and specificity. K+ uptake electrogenicity and the non-competitive inhibition due to either ATP or NADH are also shown. In the whole, the experimental findings reported in this paper demonstrate the existence of the mitochondrial K+ ATPchannel in plants (PmitoKATP). Interestingly, Mg2+ and glyburide, which can inhibit mammalian K+ channel, have no effect on PmitoKATP. In the presence of the superoxide anion producing system (xanthine plus xanthine oxidase), PmitoKATP activation was found. Moreover, an inverse relationship was found between channel activity and mitochondrial superoxide anion formation, as measured via epinephrine photometric assay. These findings strongly suggest that mitochondrial K+ uptake could be involved in plant defense mechanism against oxidative stress due to reactive oxygen species generation.

Effects of fatty acids, nucleotides and reactive oxygen species on durum wheat mitochondria.

Linoleic acid (LA) and other fatty acids added to respiring durum wheat mitochondria (DWM) were found to cause a remarkable membrane potential (ΔΨ) decrease, as monitored by measuring safranin fluorescence. The rate of ΔΨ decrease showed (i) saturation dependence on LA concentration; (ii) fatty acid specificity; (iii) inhibition by externally added ATP, GDP, GTP and Mg2+ and (iv) sigmoid dependence upon initial ΔΨ, thus suggesting the existence of an active plant mitochondrial uncoupling protein (PUMP) in mitochondria from monocotyledonous species (durum wheat, Triticum durum Desf.). Surprisingly, the rate of the linoleate dependent ΔΨ decrease was found to be activated by reactive oxygen species (ROS) (hydrogen peroxide and superoxide anion) and, moreover, linoleate proved to lower the mitochondrial generation of superoxide anion. These results suggest that ROS can activate PUMP, thus protecting the cell against mitochondrial ROS production.

Seawater stress applied at germination affects mitochondrial function in durum wheat (Triticum durum) early seedlings

Seawater stress effects on mitochondrial ATP synthesis and membrane potential (ΔΨ) were investigated in germinating durum wheat seedlings under moderate (22% seawater osmolarity, -0.62 MPa) and severe (37% seawater osmolarity, -1.04 MPa) stress. To estimate the osmotic component of salt stress, mannitol solutions (0.25 and 0.42 m) iso-osmotic with the saline ones were used. Moderate stress intensity only delayed mean germination time (MGT), whereas higher seawater osmolarity reduced germination percentage as well. In contrast, Na+ and Cl- accumulation showed a sharp increase under moderate stress and only a small further increase under severe stress, which was more pronounced for Cl-. Only severe stress significantly damaged succinate-dependent oxidative phosphorylation, which may be related to the stress-induced alteration in inner mitochondrial membrane permeability, as indicated by changes in ΔΨ profiles. Proline-dependent oxidative phosphorylation, however, was inhibited under moderate stress. This suggests the occurrence of an adaptation mechanism leading to proline accumulation as an osmoprotectant. Moreover, both the osmotic and the toxic components of seawater stress were detrimental to oxidative phosphorylation. Damage to germination and MGT, in contrast, were mainly caused by osmotic stress. Therefore, mitochondrial function appears to be a more sensitive target of toxic stress than growth. In conclusion, the effects of seawater stress on mitochondrial ATP synthesis vary in relation to the substrate oxidised and stress level, inducing both adaptive responses and damage.

Isolated Durum Wheat and Potato Cell Mitochondria Oxidize Externally Added NADH Mostly via the Malate/Oxaloacetate Shuttle with a Rate That Depends on the Carrier-Mediated Transport

We investigated whether and how mitochondria from durum wheat (Triticum durum Desf.) and potato (Solanum tuberosum), isolated from etiolated shoots and a cell suspension culture, respectively, oxidize externally added NADH via the mitochondrial shuttles; in particular, we compared the shuttles and the external NADH dehydrogenase (NADH DHExt) with respect to their capacity to oxidize external NADH. We found that external NADH and NADPH can be oxidized via two separate DHExt, whereas under conditions in which the activities of NAD(P)H DHExt are largely prevented, NADH (but not NADPH) is oxidized in the presence of external malate (MAL) and MAL dehydrogenase, in a manner sensitive to several non-penetrant compounds according to the occurrence of the MAL/oxaloacetate (OAA) shuttle. In durum wheat mitochondria and potato cell mitochondria, the rate of NADH oxidation was limited by the rate of a novel carrier, the MAL/OAA antiporter, which is different from other carriers thought to transport OAA across the mitochondrial membrane. No NAD(P)H oxidation occurred arising from the MAL/Aspartate and the α-glycerophosphate/dihydroxyacetonphosphate shuttles. We determined the kinetic parameters of the enzymes and the antiporter involved in NADH oxidation, and, on the basis of a kinetic analysis, we showed that, at low physiological NADH concentrations, oxidation via the MAL/OAA shuttle occurred with a higher efficiency than that due to the NADH DHExt (about 100- and 10-fold at 1 μm NADH in durum wheat mitochondria and in potato cell mitochondria, respectively). The NADH DHExt contribution to NADH oxidation increased with increasing NADH concentration.

Activation of the plant mitochondrial potassium channel by free fatty acids and acyl-CoA esters: a possible defence mechanism in the response to hyperosmotic stress

The effect of free fatty acids (FFAs) and acyl-CoA esters on K(+) uptake was studied in mitochondria isolated from durum wheat (Triticum durum Desf.), a species that has adapted well to the semi-arid Mediterranean area and possessing a highly active mitochondrial ATP-sensitive K(+) channel (PmitoK(ATP)), that may confer resistance to environmental stresses. This was made by swelling experiments in KCl solution under experimental conditions in which PmitoK(ATP) activity was monitored. Linoleate and other FFAs (laurate, palmitate, stearate, palmitoleate, oleate, arachidonate, and the non-physiological 1-undecanesulphonate and 5-phenylvalerate), used at a concentration (10 μM) unable to damage membranes of isolated mitochondria, stimulated K(+) uptake by about 2-4-fold. Acyl-CoAs also promoted K(+) transport to a much larger extent with respect to FFAs (about 5-12-fold). In a different experimental system based on safranin O fluorescence measurements, the dissipation of electrical membrane potential induced by K(+) uptake via PmitoK(ATP) was found to increase in the presence of 5-phenylvalerate and palmitoyl-CoA, both unable to elicit the activity of the Plant Uncoupling Protein. This result suggests a direct activation of PmitoK(ATP). Stimulation of K(+) transport by FFAs/acyl-CoAs resulted in a widespread phenomenon in plant mitochondria from different mono/dicotyledonous species (bread wheat, barley, triticale, maize, lentil, pea, and topinambur) and from different organs (root, tuber, leaf, and shoot). Finally, an increase in mitochondrial FFAs up to a content of 50 nmol mg(-1) protein, which was able to activate PmitoK(ATP) strongly, was observed under hyperosmotic stress conditions. Since PmitoK(ATP) may act against environmental/oxidative stress, its activation by FFAs/acyl-CoAs is proposed to represent a physiological defence mechanism.

Reactive oxygen species inhibit the succinate oxidation-supported generation of membrane potential in wheat mitochondria

In order to gain a first insight into the effects of reactive oxygen species (ROS) on plant mitochondria, we studied the effect of the ROS producing system consisting of xanthine plus xanthine oxidase on the rate of membrane potential (ΔΨ) generation due to either succinate or NADH addition to durum wheat mitochondria as monitored by safranin fluorescence. We show that the early ROS production inhibits the succinate‐dependent, but not the NADH‐dependent, ΔΨ generation and oxygen uptake. This inhibition appears to depend on the impairment of mitochondrial permeability to succinate. It does not involve mitochondrial thiol groups sensitive to either mersalyl or N‐ethylmaleimide and might involve both protein residues and/or membrane lipids, as suggested by the mixed nature. We propose that, during oxidative stress, early generation of ROS can affect plant mitochondria by impairing metabolite transport, thus preventing further substrate oxidation, ΔΨ generation and consequent large‐scale ROS production.

Increase of Membrane Permeability of Mitochondria Isolated from Water Stress Adapted Potato Cells

In order to gain some insight into mitochondria permeability under water stress, intact coupled mitochondria were isolated from water stress adapted potato cells and investigations were made of certain transport processes including the succinate/malate and ADP/ATP exchanges, the plant mitochondrial ATP-sensitive potassium channel (PmitoKATP) and the plant uncoupling mitochondrial protein (PUMP). The VmaxL values measured for succinate/malate and ADP/ATP carriers, as photometrically investigated, as well as the same values for the PmitoATP and the PUMP were found to increase; this suggested that mitochondria adaptation to water stress can cause an increase in the membrane permeability.

Photochemical quenching of chlorophyll fluorescence and drought tolerance in different durum wheat ( Triticum durum ) cultivars

The aim of this study was to identify a fluorescence parameter whose estimate could be used reliably for a drought tolerance test in durum wheat ( Triticum durum ). Twenty-five cultivars were grown in a glasshouse over two seasons (1987/88 and 1988/89) at Foggia, Southern Italy. Photochemical and non-photochemical quenching ( q Q and q E ), the half time of fluorescence decay ( tP ½) and the initial slope of fluorescence decay ( IS PS ) were measured on control and dehydrated pre-darkened excised leaves; q Q and q E were measured twice: first at 14 s after actinic illumination and second at the steady state. No great difference in q Q and q E was apparent between control and dehydrated leaves at the steady state; however, at 14 s after illumination there was a decrease in q Q and in IS PS and an increase in tP ½ in dehydrated leaves. The predictive capability of fluorescence parameters was assessed by comparison with a yield-based drought susceptibility index (DSI). The percentage decrease in q Q at 14 s showed the highest correlation with DSI ( r = 0·75, significant at P Fo , P and Fm ) and parameters ( q Q , q E and tP ½) indicated that for maximum reliability the test must be applied at the vegetative phase.

The maintenance of photosynthetic electron transport in relation to osmotic adjustment in durum wheat cultivars differing in drought resistance

Abstract The relationships between photosynthesis and osmotic adjustment (OA) under water stress are not yet well understood. Therefore, a possible association between the quantum yield of photosynthetic electron transport at the Photosystem II level (ΦPSII) and OA was evaluated in drought-stressed durum wheat plants in this paper. Four cultivars (cvs) differing in the degree of drought resistance as estimated by the ΦPSII maintenance, were grown in a growth chamber under two different water stress cycles; in both cases two stress intensities (moderate and severe) were used. Leaf water potential (ΨW), osmotic potential at full turgor (Ψπ100), ΦPSII and the efficiency of excitation capture by open Photosystem II reaction centres (ECPSII) were determined in the different experimental conditions, both in control and stressed plants. ΦPSII and ECPSII were evaluated by in vivo chlorophyll fluorescence analysis. ΦPSII showed a significant decrease only under severe water stress and for the drought susceptible cvs; on the other hand, ECPSII showed no change under the different water stress conditions. The percent decrease of ΦPSII under water deficit was confirmed to be a good indicator of drought resistance in durum wheat. OA was estimated as the difference in the Ψπ100 between leaves from water-stressed and well-watered plants. No relationship between the maintenance of the ΦPSII and OA under water deficit was observed in the adopted experimental conditions; this result is discussed in the light of the contrasting evidence in the literature.