Use of micro-PIXE to determine spatial distributions of copper in Brassica carinata plants exposed to CuSO4 or CuEDDS.

A better understanding of the mechanisms that govern copper (Cu) uptake, distribution and tolerance in Brassica carinata plants in the presence of chelators is needed before significant progress in chelate-assisted Cu phytoextraction can be made. The aims of this study were therefore to characterise (S,S)-N,N'-ethylenediamine disuccinic acid (EDDS)-assisted Cu uptake, and to compare the spatial distribution patterns of Cu in the roots and leaves of B. carinata plants. The plants were treated with 30 µM or 150 µM CuSO(4) or CuEDDS in hydroponic solution. Quantitative Cu distribution maps and concentration profiles across root and leaf cross-sections of the desorbed plants were obtained by micro-proton induced X-ray emission. In roots, the 30 µM treatments with both CuSO(4) and CuEDDS resulted in higher Cu concentrations in epidermal/cortical regions. At 150 µM CuSO(4), Cu was mainly accumulated in root vascular bundles, whereas with 150 µM CuEDDS, Cu was detected in endodermis and the adjacent inner cortical cell layer. Under all treatments, except with a H(+)-ATP-ase inhibitor, the Cu in leaves was localised mainly in vascular tissues. The incubation of plants with 150 µM CuEDDS enhanced metal translocation to shoots, in comparison to the corresponding CuSO(4) treatment. Inhibition of H(+)-ATPase activity resulted in reduced Cu accumulation in 30 µM CuEDDS-treated roots and 150 µM CuEDDS-treated leaves, and induced changes in Cu distribution in the leaves. This indicates that active mechanisms are involved in retaining Cu in the leaf vascular tissues, which prevent its transport to photosynthetically active tissues. The physiological significance of EDDS-assisted Cu uptake is discussed.

The influence of EDDS on the metabolic and transcriptional responses induced by copper in hydroponically grown Brassica carinata seedlings.

To improve the knowledge about the use of plants for the removal of toxic metals from contaminated soils, metabolic and transcriptional responses of Brassica carinata to different forms of copper (Cu) were studied. Two-week-old hydroponically grown seedlings were exposed for 24 h to 30 µM CuSO4 or CuEDDS. CuSO4 appeared to be more toxic than CuEDDS as roots showed higher levels of thiobarbituric acid reactive substances (TBARS) and increased relative leakage ratios (RLR), although the superoxide dismutase (SOD, EC 1.15.1.1) activity increased following both exposures. In CuSO4-exposed seedlings the higher toxicity was underlined by increased transcription of lipoxygenases (EC 1.13.11.12) and NADPH oxidases (EC 1.6.99.6) and by the higher Cu accumulation in both tissues compared to CuEDDS exposure. The presence of EDDS increased Cu translocation, which resulted 5-times higher than when exposed to CuSO4. Decreases in catalase (CAT, EC 1.11.1.6), ascorbate peroxidase (APX, EC 1.11.1.11) and glutathione reductase (GR, EC 1.6.4.2) activities together with increases of reduced glutathione (GSH) and tocopherols and a reduction of lipoic acid (LA) were observed in roots of CuSO4-exposed seedlings. On the contrary, CuEDDS exposure induced a general increase in enzyme activities and decreases in ascorbate (AsA) and tocopherol levels. In the primary leaves, in both exposures Cu differently affected the oxidative stress responses indicating that the cellular redox balance was anyway maintained. EDDS plays a crucial role in B. carinata tolerance to oxidative stress induced by Cu and might be proposed to improve the efficiency of Cu phytoextraction.

Heavy metal hyperaccumulating plants: how and why do they do it? And what makes them so interesting?

The term "hyperaccumulator" describes a number of plants that belong to distantly related families, but share the ability to grow on metalliferous soils and to accumulate extraordinarily high amounts of heavy metals in the aerial organs, far in excess of the levels found in the majority of species, without suffering phytotoxic effects. Three basic hallmarks distinguish hyperaccumulators from related non-hyperaccumulating taxa: a strongly enhanced rate of heavy metal uptake, a faster root-to-shoot translocation and a greater ability to detoxify and sequester heavy metals in leaves. An interesting breakthrough that has emerged from comparative physiological and molecular analyses of hyperaccumulators and related non-hyperaccumulators is that most key steps of hyperaccumulation rely on different regulation and expression of genes found in both kinds of plants. In particular, a determinant role in driving the uptake, translocation to leaves and, finally, sequestration in vacuoles or cell walls of great amounts of heavy metals, is played in hyperaccumulators by constitutive overexpression of genes encoding transmembrane transporters, such as members of ZIP, HMA, MATE, YSL and MTP families. Among the hypotheses proposed to explain the function of hyperaccumulation, most evidence has supported the "elemental defence" hypothesis, which states that plants hyperaccumulate heavy metals as a defence mechanism against natural enemies, such as herbivores. According to the more recent hypothesis of "joint effects", heavy metals can operate in concert with organic defensive compounds leading to enhanced plant defence overall. Heavy metal contaminated soils pose an increasing problem to human and animal health. Using plants that hyperaccumulate specific metals in cleanup efforts appeared over the last 20 years. Metal accumulating species can be used for phytoremediation (removal of contaminant from soils) or phytomining (growing plants to harvest the metals). In addition, as many of the metals that can be hyperaccumulated are also essential nutrients, food fortification and phytoremediation might be considered two sides of the same coin. An overview of literature discussing the phytoremediation capacity of hyperaccumulators to clean up soils contaminated with heavy metals and the possibility of using these plants in phytomining is presented.

Contribution of major lipophilic antioxidants to the antioxidant activity of basil extracts: an EPR study.

BACKGROUND: The present research analyses the contribution of some lipid antioxidants to the antioxidant activity of lipophilic extracts from basil by an electron paramagnetic resonance (EPR) study using the stable free radical 1,1-diphenyl-2-picrylhydrazyl (DPPH). DPPH assay is considered an easy and accurate method with regard to measuring the antioxidant activity of plant extracts and EPR has already been employed to determine antioxidant activities of lipophilic plant extracts. RESULTS: Lipid extracts were obtained from basil grown hydroponically for 20 or 35 days from sowing and in soil for 35 days from sowing. Fast and slow rate constants were distinguishable in the decay kinetics of DPPH mixture added with lipid extract. Antioxidants with kinetics characterized by a fast decay rate were tocopherols and chlorophylls, whereas those characterized by a slow decay rate were carotenoids, among which were ß-carotene, lutein, ß-apo-8'-carotenal and zeaxanthin. The 20-day hydroponically grown sample, which showed higher contents of tocopherol, chlorophyll and carotenoid molecules, was the sample endowed with the higher content of fast lipophilic antioxidants (FLA) and slow lipophilic antioxidants (SLA). CONCLUSION: The three samples showed different compositions of FLA and SLA, giving rise to different decay kinetics. Despite the differences, in all samples tocopherol contributed about 0.3% to the bulk of FLA, whereas the figure for chlorophyll was about 40%, evidencing the relevant but little-studied role of chlorophyll as an antioxidant.

Uptake and translocation of CuEDDS complexes by Brassica carinata.

The knowledge of the mechanisms that underlie metal complex uptake may lead to the development of new strategies for enhancing metal phytoextraction. As metals such as copper are actively taken up by roots, by inhibiting the proton driving force it is possible to obtain preliminary indications on the metal complex uptake mechanism. For this, Cu, EDDS, and Cu-EDDS uptake kinetics of Brassica carinata excised roots incubated in 30 and 150 microM solutions of either the metal, the chelant, and the complex were determined in the presence or not of the ATPase inhibitor vanadate. Following both Cu and CuEDDS treatments, metal uptake was negatively influenced by vanadate, whereas EDDS uptake did not, suggesting that Cu and the chelant did not enter the roots in their complexed form but by two different routes. The incubation in the same solutions of B. carinata intact plants showed that, differently from Cu, EDDS was largely translocated to shoots, but its low concentration resulted in a Cu to EDDS molar ratio ranging from 2 to 4 depending on metal complex concentration in the solution confirming that the uptake pathways of the two compounds were different.

Lipoic acid and redox status in barley plants subjected to salinity and elevated CO2.

Future environmental conditions will include elevated concentrations of salt in the soil and an elevated concentration of CO(2) in the atmosphere. Because these environmental changes will likely affect reactive oxygen species (ROS) formation and cellular antioxidant metabolism in opposite ways, we analyzed changes in cellular H(2)O(2) and non-enzymatic antioxidant metabolite [lipoic acid (LA), ascorbate (ASA), glutathione (GSH)] content induced by salt stress (0, 80, 160 or 240 mM NaCl) under ambient (350 micromol mol(-1)) or elevated (700 micromol mol(-1)) CO(2) concentrations in two barley cultivars (Hordeum vulgare L.) that differ in sensitivity to salinity (cv. Alpha is more sensitive than cv. Iranis). Under non-salinized conditions, elevated CO(2) increased LA content, while ASA and GSH content decreased. Under salinized conditions and ambient CO(2), ASA increased, while GSH and LA decreased. At 240 mM NaCl, H(2)O(2) increased in Alpha and decreased in Iranis. When salt stress was imposed at elevated CO(2), less oxidative stress and lower increases in ASA were detected, while LA was constitutively higher. The decrease in oxidative stress could have been because of less ROS formation or to a higher constitutive LA level, which might have improved regulation of ASA and GSH reductions. Iranis had a greater capacity to synthesize ASA de novo and had higher constitutive LA content than did Alpha. Therefore, we conclude that elevated CO(2) protects barley cultivars against oxidative damage. However, the magnitude of the positive effect is cultivar specific.

The effect of salinity on photosynthetic activity in potassium-deficient barley species.

The interactive effects of salinity and potassium deficiency on the growth, mineral elements and photosynthetic performance were investigated in wild (Hordeum maritimum L.) and cultivated barley (Hordeum vulgare L. var. Manel). At 28 d of growth, plants were treated with 3 mM K and 0 mM NaCl (3-0); 3 mM K and 100 mM NaCl (3-100); 0 mM K and 0 mM NaCl (0-0), 0 mM K and 100 mM NaCl (0-100) for 14 d. In both species, biomass production decreased considerably when the two constraints were applied simultaneously. Salinity affected shoots more than roots, whereas for potassium deficiency, the reverse occurred. Generally, potassium uptake was more affected in wild than in cultivated barley and, independent of potassium availability, 100 mM NaCl increased Na+ content in both species, whereas K+ deprivation increased Na(+) content only in H. maritimum shoots (0-0). Potassium-use efficiency (KUE) increased in all treated plants. Potassium deficiency increased the negative effects induced by salt in the photosynthetic process of H. vulgare, and this species seemed to be unable to counteract the negative effects of salinity. H. maritimum showed limitation in CO2 photoassimilation, but this species displayed mechanisms that play a role in avoiding PSII photodamage aimed to dissipate the excess energy.

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.

The oxidative stress caused by salinity in two barley cultivars is mitigated by elevated CO2.

Changes in antioxidant metabolism because of the effect of salinity stress (0, 80, 160 or 240 mM NaCl) on protective enzyme activities under ambient (350 micromol mol(-1)) and elevated (700 micromol mol(-1)) CO(2) concentrations were investigated in two barley cultivars (Hordeum vulgare L., cvs Alpha and Iranis). Electrolyte leakage, peroxidation, antioxidant enzyme activities [superoxide dismutase (SOD), EC 1.15.1.1; ascorbate peroxidase (APX), EC 1.11.1.11; catalase (CAT), EC 1.11.1.6; dehydroascorbate reductase (DHAR), EC 1.8.5.1; monodehydroascorbate reductase (MDHAR), EC 1.6.5.4; glutathione reductase (GR), EC 1.6.4.2] and their isoenzymatic profiles were determined. Under salinity and ambient CO(2), upregulation of antioxidant enzymes such as SOD, APX, CAT, DHAR and GR occurred. However, this upregulation was not enough to counteract all ROS formation as both ion leakage and lipid peroxidation came into play. The higher constitutive SOD and CAT activities together with a higher contribution of Cu,Zn-SOD 1 detected in Iranis might possibly contribute and make this cultivar more salt-tolerant than Alpha. Elevated CO(2) alone had no effect on the constitutive levels of antioxidant enzymes in Iranis, whereas in Alpha it induced an increase in SOD, CAT and MDHAR together with a decrease of DHAR and GR. Under combined conditions of elevated CO(2) and salinity the oxidative damage recorded was lower, above all in Alpha, together with a lower upregulation of the antioxidant system. So it can be concluded that elevated CO(2) mitigates the oxidative stress caused by salinity, involving lower ROS generation and a better maintenance of redox homeostasis as a consequence of higher assimilation rates and lower photorespiration, being the response dependent on the cultivar analysed.

Implication of phospholipase D in response of Hordeum vulgare root to short-term potassium deprivation.

To verify the possible involvement of lipids and several other compounds including hydrogen peroxide (H(2)O(2)) and glyceraldehyde-3-phosphate dehydrogenase (G3PDH) in the response of Hordeum vulgare to early potassium deprivation, plants were grown in hydroponic conditions for 30d with a modified Hewitt nutrient solution containing 3mM K(+). They were then incubated for increasing periods of time ranging from 2 to 36h in the same medium deprived of K(+). In contrast to leaves, root K(+) concentration showed its greatest decrease after 6h of treatment. The main lipids of the control barley roots were phospholipids (PL), representing more than 50% of the total lipids. PL did not change with treatment, whereas free sterols (FS) decreased following K(+) deprivation, showing a reduction of approximately 17% after 36h. With respect to the individual PL, 30h K(+) deprivation led to a reduction in phosphatidylcholine (PC), phosphatidylserine (PS), and phosphatidylinositol (PI) levels, whereas phosphatidylglycerol (PG), phosphatidylethanolamine (PE), and phosphatidic acid (PA) levels increased. The maximum PA accumulation and the highest phospholipase D (PLD) activation, estimated by an accumulation of phosphatidylbutanol (PtBut), were observed after 24h of K(+) starvation. At the root level, H(2)O(2) showed the maximum value after 6h of incubation in -K solution. In parallel, G3PDH activity reached its minimum. On the basis of a concomitant stimulation of PLD activity and, consequently, PA accumulation, enhancement of H(2)O(2) production, and inhibition of G3PDH activity, we suggest a possible involvement of these three compounds in an early response to K(+) deprivation.

Irrigation with diluted seawater improves the nutritional value of cherry tomatoes.

The aim of this study was to assess whether the nutritional value of cherry tomato can be improved by irrigating plants with diluted seawater (12%; EC = 10 mS/cm in comparison with a control at EC = 4 mS/cm). Berries of cherry tomato cv. Naomi were analyzed at the red-ripe stage for the contents of NADPH and NADP+ as well as for the amounts of the main antioxidants, such as ascorbic acid, lipoic acid, tocopherols, and phenolic acids. As compared to the controls, the fruits of salt-treated plants showed a higher titratable acidity and a higher concentration of reducing sugars. The fruits picked from tomato plants irrigated with diluted seawater produced berries characterized by a higher nutritional value. Following salinity, berries showed higher amounts of vitamin C, vitamin E, dihydrolipoic acid, and chlorogenic acid. It was hypothesized that protocatechuic, vanillic, caffeic, and ferulic acids were utilized to counteract the damaging effects of salinity-induced oxidative stress, allowing tomato fruits to maintain a high reduced status even following salinization.

Characterization of polyphenol oxidase changes induced by desiccation of Ramonda serbica leaves.

Resurrection plants are able to dehydrate/rehydrate rapidly without cell damage by a mechanism, the understanding of which may be of ecological importance in the adaptation of crop plants to dry conditions. The o-diphenol oxidase in Ramonda serbica Pan. & Petrov, a rare resurrection plant of the Balkan Peninsula, was characterized in respect to different isoforms, preferable substrates and specific inhibitors. Two anionic isoforms with pI 4.6 and 4.7 were separated from turgid leaves. Three additional anionic isoforms (pI 5.1, 5.3 and 5.6) and three neutral isoforms (pI from 6.8 to 7.4) were induced in desiccated leaves. Based on apparent K(m) values, the affinity for reducing substrates decreased as follows: methyl catechol > chlorogenic acid > 3,4-dihydroxyphenylalanine > caffeic acid > pyrogallol. Polyphenol oxidase (PPO) activity was specifically sensitive to diethyldithiocarbamate and also inhibited by KCN, DTT and salicylic hydroxamic acid but with no inhibitory effect of Na3N. Plants were subjected to drought-to-near complete water loss (approximately 2% relative water content, RWC) and several fold higher PPO activity was detected in desiccated leaves. Ramonda leaves contain high levels of phenolics, which decreased during drought. Rehydration of dry leaves from 2% RWC to 95% RWC led to transient inhibition of PPO in the first few hours. Within a day, the levels completely recovered to those determined in desiccated leaves. The finding of desiccation-induced high activity of PPO and new isoforms, which were also present in rehydrated turgid leaves, indicates a substantial role for PPO in the adaptation mechanism of resurrection plants to desiccation and also to the oxidative stress during rehydration.

Long- and short-term phosphate deprivation in bean roots: plasma membrane lipid alterations and transient stimulation of phospholipases.

In a long-term experiment bean (Phaseolus vulgaris L.) seedlings were grown for 18 days in hydroponics in either phosphate-sufficient (+P) or phosphate-deficient (-P) nutrient solutions. Phosphate deprivation halved the phosphorous content of roots. In plasma membrane (PM) fractions isolated from -P roots the phospholipid (PL) level was reduced from 35 to 21 mol%, while PL composition and degree of unsaturation were hardly altered. Digalactosyldiacylglycerol (DGDG) accumulated up to 26% of total PM lipids, replacing PL to a large extent. Molecular species and fatty acid compositions of DGDG in root PM were different compared to DGDG present in the -P plastids. In a short-term study, bean seedlings were grown for 18 days in hydroponics with a complete nutrient solution containing phosphate and then incubated in a -P medium for increasing time ranging from 1 up to 96 h. At the end of the starvation period phosphorous content of -P roots was reduced by 30% compared to +P ones. An activation of phospholipase D and phospholipase C was observed after 1 and 2h of phosphate deprivation, respectively. Maximal phosphatidic acid accumulation was detected after 4h of phosphate deprivation, when also DGDG started to accumulate in PM of bean roots. The fatty acid composition of PLD-derived phosphatidylbutanol resembled that of phosphatidylcholine.

The use of NTA and EDDS for enhanced phytoextraction of metals from a multiply contaminated soil by Brassica carinata.

The potential of nine different species to grow in the presence of metals (As, Cd, Cu, Pb and Zn) and to accumulate them in the shoots was assessed for each metal separately by germination and root length tests, and successively by hydroponic experiments. Of the nine species tested, Brassica carinata was the species that accumulated the highest amounts of metals in shoots without suffering a significant biomass reduction. To further evaluate the potential of B. carinata for chelant-enhanced phytoextraction of a natural, multiply metal-polluted soil (As, Cd, Cu, Pb and Zn), both hydroponic and pot experiments were carried out with nitrilotriacetic acid (NTA) or (S,S)-ethylenediamine disuccinic acid (EDDS) as complexing agents. The hydroponic study with solutions containing the five metals together showed that accumulation of Cd, Cu, Pb and Zn in shoots was higher following EDDS addition compared to NTA. EDDS was more effective than NTA in desorbing Cu, Pb and Zn from the soil, whereas As and Cd were poorly extracted. B. carinata plants were grown for 4 weeks in the multiply metal-contaminated soil and then the soil was amended with 5 mmol kg(-1) NTA or EDDS. All plants were harvested 1 week after amendment. In comparison to NTA, EDDS was more effective in enhancing the concentrations of Cu, Pb and Zn in B. carinata shoots (2- to 4-fold increase compared to the control). One week after chelant addition, the DTPA-extractable metal concentrations in the polluted soil were lower in the EDDS treatment in comparison with the NTA amendment. Even though B. carinata showed a reduced growth and a relatively low metal uptake, it demonstrated the ability to survive and tolerate the presence of more metals simultaneously.

Early production of activated oxygen species in root apoplast of wheat following copper excess.

Wheat seedlings (Triticum durum Desf.) were incubated in a solution containing 100 microM CuSO(4) for increasing time ranging from 1 min to 6h. Copper rapidly accumulated into the roots, and its amount increased significantly until 360 min. During the experiment, copper did not cause any lipid peroxidation and K(+) leakage. Up to 60 min of copper treatment the superoxide (O2(*-)) production in root apoplast decreased concomitantly with increase in superoxide dismutase (SOD) activity. In contrast, after 60 min of incubation, SOD decreased and this facilitated an increase in O2(*-) production. In the presence of the SOD inhibitor diethyldithiocarbamic acid, O2(*-) production was more than two times higher and showed a biphasic increase. Very high SOD activity in the apoplast, due to the presence of three different isozymes, one Mn-SOD and two CuZn-SODs, dismutated the radical giving rise, at least in part, to an increase in hydrogen peroxide. The highest value of H(2)O(2) was detected at 15 min, when peroxidase (POD) activity reached the lowest value. Root apoplast showed the presence of at least five different isoforms of PODs, whose pattern did not change during the entire treatment.

Copper excess triggers phospholipase D activity in wheat roots.

Wheat seedlings (Triticum durum Desf.) were incubated in 100muM Cu(2+) for different periods of time ranging from 1min up to 16h. Following metal addition a rapid intake of copper ions into the roots was observed. Cu(2+) induced an accumulation of both phosphatidic acid and phosphatidylbutanol within 1min of incubation, the latter indicating a very rapid induction of phospholipase D (PLD) activity. The highest PLD stimulation was detected after 2h from copper addition and decreased almost to the initial value at increasing times. Cycloheximide treatment of roots lowered phosphatidylbutanol accumulation because of a reduced PLD activity. The expression profile of a T. durum putative PLD-encoding gene showed a peak after 1h of treatment as well, indicating that enhanced gene expression contributed to the increase in PLD activity. In the absence of copper ions, roots treated with the G protein activator mastoparan showed increases in phosphatidic acid and phosphatidylbutanol similar to those detected with the metal. PLD activity was also stimulated by cholera toxin. Two putatively G protein alpha subunit encoding sequences were isolated and no significant differences in transcription activity following Cu(2+) addition were observed. In copper-treated roots an early production of superoxide generated both by total and membrane-bound NADPH oxidase occurred. The G protein inhibitor suramin as well as the PLD antagonist 1-butanol abolished copper-induced superoxide production.

Influence of sodium nitrilotriacetate (NTA) and citric acid on phenolic and organic acids in Brassica juncea grown in excess of cadmium.

Brassica juncea cv. 426308 was grown in soils containing 150 mg Cd(2+)kg(-1) soil. After 38 days, the soil was amended with two rates of citric acid or NTA (10 and 20 mmol kg(-1) soil). Control soil was not amended with chelates. Plants were harvested during growth, immediately before and seven days after chelate addition. Shoot composition of organic and phenolic acids and shoot Cd(2+) concentration were determined. Cadmium concentration remained constant during the growth and increased following NTA and citric acid amendments depending on chelate type and concentration. The highest increments in Cd(2+) were measured after the addition of NTA. Compared to the control, 10 and 20 NTA-treated plants showed two- and three-fold increases, respectively. At 150 mg Cd(2+)kg(-1) soil the amount of organic and phenolic acids in the leaves of B. juncea was always higher than that detected in the control. A direct correlation between organic acid concentration and cadmium content was detected both during growth and after chelate addition. On the contrary, the amount of phenols seemed to be correlated with the metal content only in non-amended and NTA-treated plants. The 10 and 20 citric acid additions caused 45% and 90% increases in shoot phenolic acids although cadmium content rose to a smaller extent. The inhibition of citrate synthase and the entrance of phosphoenolpyruvate in shikimate pathway leading to the formation of aromatic compounds might come into play. The increase in phenylalanine ammonialyase activity following citric acid amendments suggested this metabolic response.

Senescence- and drought-related changes in peroxidase and superoxide dismutase isoforms in leaves of Ramonda serbica.

Ramonda sp. (Gesneriaceae) is an endemic and relic plant in a very small group of poikilohydric angiosperms that are able to survive in an almost completely dehydrated state. Senescence- and drought-related changes in the activity of peroxidase (POD; EC 1.11.1.7), ascorbate peroxidase (EC 1.11.1.11), and superoxide dismutase (SOD; EC 1.15.1.1) were determined in leaves of different age and relative water content. The results indicate that different POD isoforms were stimulated during senescence and dehydration. Two of the soluble POD isoforms were anionic with pI 4.5, and two were cationic with pI 9.3 and 9.0. The activity of ascorbate peroxidase remained unchanged either by drought or senescence. For the first time, SOD isoforms have now been determined in this resurrection plant. Several SOD isoforms, all of the Mn type, were found to be anionic with pI 4 and a few others had pI from 5 to 6, while one band of FeSOD with a lower molecular weight was neutral. Rehydration brought about a remarkable decrease over the first hour in the activity of all the antioxidant enzymes examined but activity recovered 1 d after rehydration. The results confirmed that dehydration and senescence caused disturbance in the redox homeostasis of Ramonda leaves, while inducing different POD isoforms. A physiological role of peroxidase reaction with hydroxycinnamic acids in conservation and protection of cellular constituents of desiccated Ramonda leaves is suggested.

Antioxidative responses of Calendula officinalis under salinity conditions.

To gain a better insight into long-term salt-induced oxidative stress, some physiological parameters in marigold (Calendula officinalis L.) under 0, 50 and 100 mM NaCl were investigated. Salinity affected most of the considered parameters. High salinity caused reduction in growth parameters, lipid peroxidation and hydrogen peroxide accumulation. Under high salinity stress, a decrease in total glutathione and an increase in total ascorbate (AsA + DHA), accompanied with enhanced glutathione reductase (GR, EC 1.6.4.2) and ascorbate peroxidase (APX, EC 1.11.1.11) activities, were observed in leaves. In addition, salinity induced a decrease in superoxide dismutase (SOD, EC 1.15.1.1) and peroxidase (POX, EC 1.11.1.7) activities. The decrease in dehydroascorbate reductase (DHAR, EC 1.8.5.1) and monodehydroascorbate reductase (MDHAR, EC 1.6.5.4) activities suggests that other mechanisms play a major role in the regeneration of reduced ascorbate. The changes in catalase (CAT, EC 1.11.1.6) activities, both in roots and in leaves, may be important in H2O2 homeostasis.

The role of lipoic acid in the regulation of the redox status of wheat irrigated with 20% sea water.

The effect of irrigation with 20% sea water was studied in 14 and 21-day-old seedlings of durum wheat (Triticum durum, cv. Ofanto). Comparisons between control (Hoagland's 2 solution) and treated (20% sea water in Hoagland's solution) plants included, besides HPLC determination of reduced (DHLA) and oxidised (LA) forms of lipoic acid, ascorbate and glutathione contents, their redox status, the activity of ascorbate peroxidase (APX, EC 1.11.1.11.) and glutathione reductase (GR, EC 1.6.4.2.). The results indicated a more relevant presence of lipoic acid in the roots in comparison to the shoots. An involvement of its reduced form in the regeneration of the reduced glutathione, at least at 14 days of treatment, suggested, besides its role as dehydrogenase enzyme cofactor, a role in the recycling of the other antioxidants. The amount of LA always increased with growth in shoots and decreased in roots, while DHLA remained constant in control and increased in treated plants. Besides, the oxidised form always decreased with sea water while the reduced form decreased in shoots and increased in roots. The ascorbate pool exerted its positive influence especially in the shoots, while APX and GR activities resulted differently modulated by the salinity level.