Mike F. Quartacci

Lipoic acid: a unique antioxidant in the detoxification of activated oxygen species

Lipoic acid (1,2-dithiolane-pentanoic acid) is a dithiol which is effective in affording protection against oxidative stress by virtue of its two sulphydryl moieties. It is present in all kinds of eukaryotic and prokaryotic cells. As lipoamide, it functions as a cofactor in the multienzyme complexes that catalyse the oxidative decarboxylation of α-keto acids such as pyruvate, α-ketoglutarate, and branched-chain α-keto acids. The complete enzyme pathway responsible for the de novo synthesis of lipoic acid has not yet been elucidated. Octanoic acid appears to be the precursor for the eight-carbon fatty acid chain, and cysteine the source of sulfur. Lipoic acid is unique, among antioxidants, because it retains powerful antioxidant properties in both its reduced (dihydrolipoic acid) and oxidised (lipoic acid) forms. Both lipoic and dihydrolipoic acids have metal-chelating ability and quench activated oxygen species either in the cytosol or in the hydrophobic domains. Dihydrolipoic acid has more antioxidant properties than lipoic acid, and it plays an important role in the recycling of other oxidised radical scavengers such as glutathione, ascorbate and tocopherol. However, dihydrolipoic acid can also exert pro-oxidant properties both by its iron-reducing ability and by its ability to generate sulfur-containing radicals that can damage proteins. There are few quantitative data on lipoic acid contents in vegetables. It has been found in asparagus, wheat and potatoes, and recently, the presence of both lipoic and dihydrolipoic acids in roots, leaves and in the stroma of wheat has been demonstrated.

Water Stress and Free Radical Mediated Changes in Sunflower Seedlings

Summary Sunflower seedlings were grown in well watered and dryland conditions obtained by withholding water. In the stressed seedlings the leaf water potential was 1.0 MP a lower than the control and the relative water content decreased as well. The reduction in the osmotic potential at full turgor following water stress indicated that an osmotic adjustment came into play. Water deficit conditions resulted in reduced growth of the plants and in increased membrane permeability. In comparison to the control, the unwatered seedlings showed lower chlorophyll and soluble protein amounts, as well as a reduction in total and polar lipid contents. Neither decrease in the unsaturation of lipids nor increase in the malondialdehyde level were observed upon water stress. In the sunflower subjected to drought, ESR spectra showed a 17 % higher free radical production; moreover, reduced superoxide dismutase (EC 1.15.1.1) and catalase (EC 1.11.1.6) activities were also found.

Growth and mineral absorption in maize seedlings as affected by increasing NaCl concentrations

Abstract The influence of NaCl solutions of decreasing osmotic potentials (¥π = ‐0.44 and ‐0.88 MPa) on seedling growth and on the concentration of the most important macro‐ and micro‐nutrients in the shoots and roots of maize (Zea mays L., cv. Summer II) grown in Hoaglands solution in a growth chamber was studied. Salt stress was imposed on six‐day‐old seedlings for a three day period. Increasing NaCl concentrations induced a reduction in the leaf water potential and a significant decrease in the length and dry weight of the shoots, whereas these two parameters decreased in the roots only at the lowest osmotic potential. Although the absorption and accumulation of nutrients upon salt stress differed in the two treatments depending on the plant tissue and nutrient, almost all of the macronutrients decreased in the roots and shoots, showing the lowest values at ¥π = ‐0.88 MPa. Sodium and Cl increased continuously, much more in the roots than in the shoots. A different response was seen in the shoots and r...

Lipid Composition and Protein Dynamics in Thylakoids of Two Wheat Cultivars Differently Sensitive to Drought

Two wheat (Triticum durum Desf.) cultivars with different sensitivities to drought were either grown under regular irrigation or subjected to water deficit by withholding water for 14 d. Water-stressed plants of both cultivars underwent similar decreases in leaf water potential, but the drought-tolerant cultivar showed higher relative water content and turgor. Neither osmotic nor elastic adjustment mechanisms appeared to be active under the conditions described here. Thylakoids isolated from the stressed, drought-tolerant wheat showed an increase in lipid-to-protein ratio, in comparison with the control, whereas this ratio remained unchanged in the sensitive wheat. In both cultivars, water deficit determined different rearrangements in the composition of the thylakoid individual polar lipids, but their unsaturation level remained unaffected with the exception of monogalactosyldiacylglycerol. In the drought-sensitive cultivar, an accumulation of free fatty acids together with a reduction in polar lipid amount was observed. Electron paramagnetic resonance measurements of spin-labeled proteins of stressed plants from the sensitive cv Adamello showed a higher spin label rotational correlation time together with lower sulphydryl group and mobile proteic portion levels, in comparison with the control. In the tolerant cv Ofanto, the first two parameters changed to a lesser extent following water depletion, and the mobile proteic portion was not altered.

Relation between lipoic acid and cell redox status in wheat grown in excess copper

Cell redox status and lipoic acid contents were analysed in wheat (Triticum durum Desf. cv. Creso) plants treated with 150 μM Cu to elucidate the role of the antioxidant lipoic acid against oxidative stress. In comparison with shoots, roots suffered a higher oxidative stress showing a decrease in NADPH contents and an oxidation of glutathione and ascorbate. Shoots did not evidence a clear oxidative damage since Cu was translocated in small amounts. Lipoic acid as reduced (DHLA) or oxidised (LA) form was present in both leaves and roots of wheat. Analysis of the cell sap showed that this antioxidant was present also as free form. The analyses showed that stroma contained significant amounts of free LA and that, after acidic hydrolysis, higher amounts of LA and DHLA were released. However, lipoic acid was undetectable in both thylakoids and microsomal membranes. Cu treatment did not determine changes in the contents of total LA and DHLA in roots, they being likely involved in Cu chelation. In contrast, in leaves after 48 h of treatment the metal induced an increase in DHLA, which could in part explain the reduction in the oxidised glutathione levels. In leaves free lipoic acid was more prone to be oxidised compared to the bound form, and the reduced form disappeared in both leaves and roots after Cu treatment.

Superoxide and hydroxyl radical generation, and superoxide dismutase in PSII membrane fragments from wheat

Illumination of photosystem II (PSII) membrane fragments of wheat (Triticum durum Desf. cv. Adamello) gave rise to both O2*- and *OH radicals adducts of the novel spin trap 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide (DEPMPO). With time, *OH became predominant displaying the conversion of O2*- into *OH. An intrinsic activity of superoxide dismutase (SOD) was found in PSII. Photoreduction of nitroblue tetrazolium (NBT) by PSII membrane fragments was induced by the addition of sodium azide and hydrogen peroxide. Western blotting of PSII proteins showed that a 29 kDa protein was recognised by an antibody against chloroplastic Fe-SOD from water lily. An increased formation rate of O2*- was observed in damaged PSII where the SOD activity decreased following a treatment with a free radical-generating system. Damage in PSII consisted also in a decrease in chlorophyll and in carotenoids as well as in a change in the lipid : chlorophyll : protein ratio. Upon treatment a decrease in the unsaturation of PSII membrane fragments was also monitored together with a degradation towards more saturated molecular species of monogalactosyldiacylglycerol.

Naturally-assisted metal phytoextraction by Brassica carinata: role of root exudates.

Due to relatively high chelant dosages and potential environmental risks it is necessary to explore different approaches in the remediation of metal-contaminated soils. The present study focussed on the removal of metals (As, Cd, Cu, Pb and Zn) from a multiple metal-contaminated soil by growing Brassica carinata plants in succession to spontaneous metallicolous populations of Pinus pinaster, Plantago lanceolata and Silene paradoxa. The results showed that the growth of the metallicolous populations increased the extractable metal levels in the soil, which resulted in a higher accumulation of metals in the above-ground parts of B. carinata. Root exudates of the three metallicolous species were analysed to elucidate their possible role in the enhanced metal availability. The presence of metals stimulated the exudation of organic and phenolic acids as well as flavonoids. It was suggested that root exudates played an important role in solubilising metals in soil and in favouring their uptake by roots.

Interactions between drought and elevated CO2 on alfalfa plants

Summary Alfalfa ( Medicago sativa L.) plants were grown in open top chambers at ambient (340 ppm) and high (600 ppm) CO 2 concentrations. Twenty-five days after the first cutting one set of both plants was subjected to water deficit conditions by withholding water for 5 days. A chamber effect on proteolytic activity, monogalactosyl diacylglycerol to digalactosyl diacylglycerol molar ratio, total non-structural carbohydrates and soluble protein contents occurred. In contrast, no change in leaf water potential was observed between plants grown outdoors and inside the chambers. Plants grown at high CO 2 concentration showed a lower decrease in leaf water potential in comparison with plants grown at atmospheric CO 2 when subjected to water stress. Under high CO 2 concentration leaf nitrogen content decreased whereas starch accumulation and a higher proteolytic activity were recorded. Following water depletion, CO 2 -enriched plants showed a decrease in total non-structural carbohydrates and soluble proteins. In thylakoid membranes high CO 2 caused an increase in chlorophyll and lipid contents and a degradation of monogalactosyl diacylglycerol. A higher degree of unsaturation in the main thylakoid lipids was also observed. CO 2 -enriched plants were less affected by water stress as shown by reduced chlorophyll degradation and a higher membrane stability.

Lipids of soybean and sunflower seedlings grown under drought conditions

Abstract Soybean and sunflower seedlings were grown under field irrigation and water deficit conditions. One set of plants was submitted to water depletion 12 days after sowing and all seedlings were harvested 20 days after sowing. In comparison to controls, the decrease in the soil water potential to - 1.5 MPa produced in both of the stressed species a reduction in leaf water potential with a consequent increase in the water saturation deficit, which was higher in sunflower. Only sunflower suffered a reduction in height, fr. wt, dry wt and in the lipid extract and lipid phosphorus content. Phospholipids decreased by more than 50% in stressed sunflower seedlings while they remained constant in soybean. Diacylglycerol and triacylglycerol concentrations increased in both species under water stress. The free sterol/phospholipid molar ratio was higher in the stressed seedlings of both species. The glycolipid content increased in soybean but was reduced in sunflower plants, with the monogalactosyldiacilglycerol being present in the largest amounts. The degree of unsaturation of the fatty acids of the various lipid classes in general did not change in response to water stress, with the exception of the diacylglycerols where it decreased. In all the lipid classes examined, unsaturation was higher than 60% in both stressed and unstressed seedlings. The free sterols presented a contrasting behaviour, showing an increase in stressed soybean and a reduction in stressed sunflower. Under water deficit conditions there was no change in the ‘more planar’ to ‘less planar’ sterol ratio.

Alterations of wheat root plasma membrane lipid composition induced by copper stress result in changed physicochemical properties of plasma membrane lipid vesicles

A response when wheat is grown in excess copper is an altered lipid composition of the root plasma membrane (PM). With detailed characterisation of the root PM lipid composition of the copper-treated plants as a basis, in the present study, model systems were used to gain a wider understanding about membrane behaviour, and the impact of a changed lipid composition.PMs from root cells of plants grown in excess copper (50 microM Cu(2+)) and control (0.3 microM Cu(2+)) were isolated using the two-phase partitioning method. Membrane vesicles were prepared of total lipids extracts from the isolated PMs, and also reference vesicles of phosphatidylcholine (PC). In a series of tests, the vesicle permeability for glucose and for protons was analysed. The vesicles show that copper stress reduced the permeability for glucose of the lipid bilayer barrier. When vesicles from stressed plants were modified by addition of lipids to resemble vesicles from control plants, the permeability for glucose was very similar to that of vesicles from control plants. The permeability for protons did not change upon stress. Electron paramagnetic resonance (EPR) of the lipid vesicles spin probed with n-doxylstearic acid (nDSA) was used to explore the lipid rotational freedom at different depth of the bilayer. The EPR measurements supported the permeability data, indicating that the copper stress resulted in more tightly packed bilayers of the PMs with reduced acyl chain motion.