Journal of Experimental Botany, Vol. 52, No. 354, pp. 77-84,
January 2001
© 2001 Oxford University Press
Original Papers |
Lipids and NADPH-dependent superoxide production in plasma membrane vesicles from roots of wheat grown under copper deficiency or excess
Dipartimento di Chimica e Biotecnologie Agrarie, Università degli Studi di Pisa, Via del Borghetto, 80, 56124 Pisa, Italy
Received 20 December 1999; Accepted 18 August 2000
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Abstract |
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The effects of in vivo copper on the lipid composition of root plasma membrane and the activities of membrane-bound enzymes, such as NADPH-dependent oxidases and lipoxygenase, were studied. Plants were grown in hydroponic culture for 11 d without Cu supply or in the presence of 50 µM Cu. Control plants were supplied with 0.3 µM Cu. Growth of roots was severely affected in the 50 µM Cu-grown plants, whereas roots grown in Cu-deficient solution did not show any difference in comparison with the control. The 50 µM Cu concentration caused an increase in the leakage of K+ ions as well. Excess metal supply resulted in a decrease in the total lipid content of plasma membrane, a higher phospholipid amount and a reduction of steryl lipids (free sterols, steryl glycosides and acylated steryl glycosides). Cu depletion in the growth solution had only a slight effect on the plasma membrane lipid composition. In comparison with the control, only the excess of Cu caused a decrease in the lipid to protein ratio as well as a change in the phospholipid composition, with a lower phosphatidylcholine to phosphatidylethanolamine ratio. The degree of unsaturation of root plasma membranes decreased following the 0 Cu treatment and even more after the 50 µM Cu supply. Plasma membranes of wheat grown under metal deficiency and excess showed increased NADPH-dependent superoxide-producing oxidase activities, whereas membrane-bound lipoxygenase was not increased or activated due to Cu treatments. The consequences of changes in plasma membrane lipid composition and activated oxygen production as a result of Cu treatments are discussed.
Key words: Copper, lipids, plasma membrane, superoxide, Triticum durum.
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Introduction |
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Both the lack and excess of copper in biotic systems is a potential hazard for plants. Cu is known to be an essential micronutrient for the development of plants, but when it accumulates to levels that exceed cellular needs Cu can be extremely toxic due to its ability to catalyse the formation of harmful free radicals or to initiate lipid peroxidation (Halliwell and Gutteridge, 1984
).
In spite of the known action of Cu on photosynthetic membranes (Droppa et al., 1987; Ciscato et al., 1997; Navari-Izzo et al., 1998; Quartacci et al., 2000), there is a lack of information regarding Cu effects at the plasma membrane (PM) level, and on its lipid composition in particular. Changes in PM integrity, resulting from alterations in the lipid composition, have been observed in plants treated with metals such as Cd, Ni (Hernandez and Cooke, 1997; Ros et al., 1992) and Al (Zhang et al., 1997).
Plant cell membranes are dynamic in behaviour, with a lipid composition changing with variations in the environment. The PM is the first functional structure in contact with toxic metals and is thought to play a critical role in plant metal tolerance. In fact, PM lipid composition controls membrane permeability and its efficiency as a semi-permeable barrier (Meharg, 1993). The PM is not protected by the intracellular detoxification mechanisms and may be injured by ions such as Cu2+. Copper is known to damage membranes due to a number of actions, including oxidation and cross-linking of protein thiols and Cu2+-catalysed production of free radicals, which initiate peroxidation of polyunsaturated fatty acids (De Vos et al., 1993). Changes in membrane lipid composition may affect the fluidity and also the intrinsic-membrane protein activities as a result of alterations in the lipidic environment in which they are embedded (Quartacci et al., 1995, 2000).
Copper-induced damage can be considered an oxidative stress mediated by activated oxygen species (Navari-Izzo et al., 1998, 1999). The production of reactive oxygen species in plants may result from the activity of redox enzymes bound or associated with the PM of the cell in addition to those associated with the electron transport systems. Different classes of enzymes are thought to contribute to this action, including lipoxygenases (LOX) and superoxide (
)-generating NAD(P)H-oxidoreductases (Van Gestelen et al., 1997). Plasma membrane-bound NAD(P)H oxidases are widely accepted as responsible for reactive oxygen species production in the regulation of defence strategies upon infection with pathogens or stimulation by elicitors (Mehdy et al., 1996). Plasma membrane oxidases use cytosolic NAD(P)H to reduce oxygen at the apoplastic membrane face. The so formed plays a major role in the oxidative burst and in other defence mechanisms, including lignin formation and phytoalexin production (Mithöfer et al., 1997). Several studies have supported the hypothesis for a role of LOX in the loss of membrane integrity following stress conditions (Siedow, 1991). Lipoxygenase can efficiently start the peroxidative breakdown of both polyunsaturated free fatty acids and complex lipids, and/or generate reactive oxygen species that can increase the deterioration and permeability of membranes (Thompson et al., 1998).
The aim of this study was to determine the changes in the lipid composition of wheat root PM isolated from plants grown under Cu deficiency or excess, and to relate the lipid alterations to the activity of superoxide-producing enzymes. To our knowledge the present study is the first to characterize the changes of wheat root PM lipids and the NADPH-dependent superoxide formation as a consequence of Cu treatments.
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Materials and methods |
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Plant material
Wheat seedlings (Triticum durum Desf. cv. Creso) were grown in hydroponic culture with continuous aeration in a growth chamber with day/night temperatures of 21/16 °C, a 16 h photoperiod, a photon flux density of 400 µmol m-2 s-1 and 70–75% RH. Seeds were surface-sterilized for 10 min with NaClO (about 2% of active chlorine), rinsed in distilled water and imbibed for 16 h with running tap water. The seeds were then placed on a floating layer of Perlite in plastic pots containing 7.5 l of a half-strength aerated Hoagland's no. 2 solution renewed every 3 d (Hoagland and Arnon, 1950
). For nutrient solution preparation precautions were taken in order to avoid the use of salts containing Cu as contaminant. Copper concentration in the nutrient solution was determined daily by atomic absorption spectrophotometry and thereafter adjusted to the initial levels by adding CuSO4. In the Hoagland's solution FeEDTA was replaced with FeSO4 and tartaric acid to avoid the formation of non-toxic CuEDTA complexes (Guo and Marschner, 1995). Plants were continuously grown with either nutrient solution containing no Cu, 0.3 (control) or 50 µM Cu (CuSO4) for 11 d. Roots were then harvested, rinsed with cold water and used immediately for leakage measurement and PM preparation, or lyophilized for Cu determination.
Leakage of K+ ions
Potassium leakage was measured by a modification of the procedure described previously (Navari-Izzo et al., 1989). The whole root systems of seedlings from each treatment were excised and washed twice to remove the contents of the cut cells. Roots were incubated in 25 ml of double distilled water and shaken at 21 °C for 24 h, and aliquots for K+ determination were taken. Roots were then immersed for 5 min in liquid N2 and placed in the same vial containing the leachate and shaken for an additional hour prior to the measurements of total K+ leached from the killed cells. Potassium released was measured by atomic absorption spectrophotometry with a hollow-cathode lamp.
Determination of Cu content
Lyophilized roots were wet-digested with concentrated HNO3, and Cu content was determined by atomic absorption spectrophotometry.
Isolation of plasma membrane
Plasma membranes were isolated using the two-phase aqueous polymer partition system. Roots were cut into pieces and immediately ground using a Braun blender in 2 vols of an extraction medium consisting of 50 mM TRIS-HCl, pH 7.5, 0.25 M sucrose, 3 mM Na2EDTA, 10 mM ascorbic acid, and 5 mM diethyldithiocarbamic acid. The homogenate was filtered through four layers of a nylon cloth and centrifuged at 10 000 g for 10 min. The supernatant was further centrifuged at 65 000 g for 30 min to yield a microsomal pellet, which was resuspended in 2 ml of a resuspension buffer (5 mM K-phosphate, pH 7.8, 0.25 M sucrose and 3 mM KCl). The plasma membrane fraction was isolated by loading the microsomal suspension (1 g) onto an aqueous two-phase polymer system to give a final composition of 6.5% (w/w) Dextran T500, 6.5% (w/w) polyethylene glycol, 5 mM K-phosphate (pH 7.8), 0.25 M sucrose, and 3 mM KCl. The PM was further purified using a two-step batch procedure. The resulting upper phase was diluted 4-fold with 50 mM TRIS-HCl, pH 7.5, containing 0.25 M sucrose, and centrifuged for 30 min at 100 000 g. The resultant PM pellet was resuspended in the same buffer containing 30% polyethylene glycol and stored at -80 °C for lipid analyses. All steps of the isolation procedure were carried out at 4 °C. Plasma membrane pellets for enzyme activity determinations were used immediately.
In order to check the purity of the PM, the activity of the vanadate-sensitive ATPase as a marker enzyme was determined (Navari-Izzo et al., 1993). Cytochrome c oxidase, NADH cytochrome c reductase and 
-sensitive ATPase activities were used as markers of mitochondria, endoplasmic reticulum and tonoplast, respectively (Navari-Izzo et al., 1993). Tests with the markers showed that, as a mean value of the isolations performed, the specific activity of ATPase was 60% higher in the PM than in the microsomal fraction; vanadate inhibited ATPase activity by 87% in the PM fraction and 36% in the microsomal one. The addition of KNO3 negligibly reduced the ATPase activity in the PM fraction (6% inhibition). The specific activities of marker enzymes such as Cyt c oxidase and NADH Cyt c reductase in the upper phase were 5% and 8%, respectively, of those determined in the lower phase. In addition, chlorophyll was not detected in the PM fraction. However, the partition behaviour of both PM and intracellular membranes may be influenced by Cu-induced alterations since the net charge density of membranes is related also to their polar head group composition. The protein content was determined taking aliquots of the PM suspension.
The analysis was performed according to Bradford with bovine serum albumin as a standard (Bradford, 1976).
Lipid extraction and separation
Lipids were extracted from the PM suspension by addition of boiling isopropanol followed by chloroform:methanol (2:1 v/v) containing butylhydroxytoluol (50 µg ml-1) as an antioxidant. The solvent mixture was then washed with 0.88% KCl to separate the chloroform phase. The upper water phase was re-extracted with choroform, the chloroform phases combined and dried under a stream of N2. The lipid extracts were stored at –20 °C and retained for further separation. Lipids were fractionated into neutral lipid, glycolipid and phospholipid (PL) fractions on Sep-Pak cartridges (Waters) (Uemura and Steponkus, 1994). Lipid extracts dissolved in chloroform:acetic acid (100:1 v/v) were transferred to the Sep-Pack cartridge and sequentially eluted with 20 ml of chloroform:acetic acid (100:1 v/v) for neutral lipids, 10 ml of acetone and 10 ml of acetone:acetic acid (100:1 v/v) for glycolipids and 7.5 ml of methanol:chloroform:water (100:50:40 by vol) for phospholipids. Chloroform (2.25 ml) and water (3 ml) were added successively to the eluate containing the PLs to obtain a phase separation and to facilitate their recovery. Separation of individual lipids was performed by TLC (Silica Gel 60, 0.25 mm thickness; Merck) with the following solvent mixtures: petroleum ether:ethyl ether:acetic acid (80:35:1 by vol) for neutral lipids (free sterols and sterol esters); chloroform:methanol: water (65:25:4 by vol) for glycolipids (steryl glycosides and cerebrosides); chloroform:methanol:acetic acid:water (85:15:10:3.5 by vol) for phospholipids. After development, bands were located with iodine vapours or spraying the plates with 0.1% Rhodamine 6G in ethanol. Individual lipids were identified by co-chromatography with authentic standards.
Quantification of lipids after TLC
Quantitative analyses of sterols, cerebrosides and phospholipids were performed as reported earlier (Navari-Izzo et al., 1993) using cholesterol, glucose and KH2PO4 as standards, respectively. All procedures were performed in the presence of silica gel from TLC.
Sterol analysis
Individual sterol components were separated and quantified by GLC as underivatized residues. Determination of acylated steryl glycosides (ASG) and steryl glycosides (SG) required chemical decomposition into sterol, acyl and sugar moieties before GLC analysis. After separation by TLC and elution from the silica gel, ASG and SG were hydrolysed with 2 N methanolic HCl under reflux for 4 h at 100 °C (Uemura and Steponkus, 1994). The resultant free sterols (FS)—and fatty acid methyl esters in ASG—were extracted using petroleum ether and analysed by GLC. The sterol moieties dissolved in ethyl acetate were analysed with a Perkin-Elmer Sigma 2B gas chromatograph using a flame ionization detector and a 30 mx0.32 mm SPB-5 fused silica capillary column (Supelco). The operating conditions were: column temperature 250 °C, injector and detector temperatures 280 °C, N2 was the carrier gas at 1 ml min-1 (split ratio 1:70). Compound identification was made on the basis of the retention time relative to known standards. Cholestane was the internal standard, and corrections were made for differences in detector response.
Fatty acid analysis
The fatty acid methyl ester derivatives from PL were obtained as previously described (Quartacci et al., 1997) and separated by GLC on a Dani 86.10 HT gas chromatograph equipped with a 60 mx0.32 mm SP-2340 fused silica capillary column (Supelco) coupled to a flame ionization detector (column temperature 175 °C). Both the injector and detector were maintained at 250 °C. Nitrogen was used as the carrier gas at 0.9 ml min-1 with a split injector system (split ratio 1:100). Heptadecanoic acid was used as the internal standard. The double bond index (DBI) was calculated as:
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NADPH-dependent 
determination
The determination of the NADPH-dependent -generating activity in isolated PM vesicles was carried out as described earlier (Van Gestelen et al., 1997), by measuring the rate of superoxide dismutase-inhibitable reduction of nitroblue tetrazolium using NADPH as electron donor. The reaction mixture consisted of 50 mM TRIS-HCl buffer, pH 7.5, 0.25 M sucrose, 0.1 mM nitro blue tetrazolium, and 50–100 µg proteins. After 1 min pre-incubation the reaction was started by the addition of 0.1 mM NADPH and the absorbance changes at 530 nm were followed for 5 min. Rates of generation were calculated using an extinction coefficient of 12.8 mM-1 cm-1.
Lipoxygenase assay
Membrane-bound LOX activity was measured as conjugated diene formation (Macrì et al., 1994). The incubation mixture contained 1.6 mM linoleic acid and 0.5% (v/v) Tween 20 in 50 mM TRIS-HCl buffer, pH 8.6. An aliquot of PM suspension containing 50–100 µg proteins was added to 2 ml reagent in the cuvette and diene formation was followed as increase of absorbance at 234 nm. An extinction coefficient of 25 mM-1 cm-1 was used to convert absorbance values to nmol of conjugated dienes (hydroperoxy linoleic acid).
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Results |
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The root length of wheat grown with excess Cu suffered a dramatic reduction (–70%) in comparison with control and plants grown in Cu-deficient solution, which did not show any change (Fig. 1A
). The Cu content of roots following the 50 µM supply increased more than 7-fold compared with control plants, whereas 0 Cu-grown roots showed a decreased amount of the metal (Fig. 1B). As shown in Fig. 1C, control plants and plants grown under Cu deficiency did not show any difference in the K+ leakage from roots. Toxicity was evident at 50 µM Cu, as indicated by the doubling of the percentage of K+ released from the whole root system in comparison with total K+ after liquid N2 treatment.
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0.05 level.The main lipids of wheat root PM were PL (Table 1
), which, in the control plants, accounted for about half of the total lipids. The other lipids found were steryl lipids (38.5%) and CER (9.2%). Among steryl lipids FS, ASG and SG were present with FS representing the predominant one (about 75%). Trace amounts of sterol esters were also detected (data not shown). As a consequence of the changes in the various classes, the total lipid amount of PM following the 50 µM Cu treatment decreased by 2.5-fold in comparison with the control and plants grown under Cu deficiency (Table 1). Copper excess caused a remarkable decrease in the amount of all the lipid classes in comparison with the control (-54% for PL, -69% for steryl lipids and –60% for CER), whereas Cu deficiency had a significant effect only on PL (+17%) and ASG (-50%). The PL to FS molar ratio showed an increase following Cu deficiency and even more so after the 50 µM Cu supply. The lipid to protein ratio was reduced only in the plants grown under metal excess (Table 1).
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The predominant PM phospholipids were phosphatidylethanolamine (PE) and phosphatidylcholine (PC) with lesser amounts of phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylserine (PS), and phosphatidic acid (PA) (Table 2
). Copper deficiency did not cause any alteration in the levels of the individual PL in comparison with the control roots. The 50 µM Cu supply resulted in a 2-fold reduction in the PC level and in an increase in PE. The amount of PA showed a significant increase as well (Table 2). The changes in the PL composition due to excess copper lowered the PC/PE ratio from 0.68 to 0.27 (Table 2).
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With regard to the steryl lipid composition (Table 3
), in the FS class the 50 µM Cu treatment determined an increase in the sitosterol level at the expense of campesterol, while the deficiency of Cu caused a decrease in cholesterol. The conjugated sterols ASG and SG showed only slight changes in the stigmasterol levels following the treatments.
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The main fatty acids of PL and ASG were palmitic and linoleic acids (Table 4
). The 50 µM Cu reduced the double bond index (DBI) in the individual PL components, with the exception of PI, as a consequence of linoleic and linolenic acid degradations. Copper deficiency caused different responses in the DBI of individual PL (Table 4). As regards the total PL, both Cu deficiency and excess induced a higher saturation in the PM fatty acids in comparison with the control plants. The ASG did not show any alteration in the unsaturation level following the treatments (Table 4).
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The activity of NADPH-dependent
-generating oxidases was affected by either the lack or excess of Cu in the nutrient solution. Compared to the controls, the production increased by 32% and 66% in the 0 and 50 µM Cu-grown roots, respectively (Fig. 2A). The membrane-bound LOX activity was unchanged due to the Cu deficiency, but was reduced in the PM isolated from roots grown at 50 µM Cu (Fig. 2B).
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Discussion |
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Both Cu deficiency and excess were asymptomatic and no visible signs of necrosis or chlorosis were observed on the leaves and roots during the treatments. The reduction in the Cu content of roots grown in the metal-deficient nutrient solution (Fig. 1B
) is an indication that the seedlings experienced Cu deficiency, but were not yet actively utilizing the ions stored in the seeds at the harvesting time. The drastic reduction in root growth at 50 µM Cu (Fig. 1A) was associated with a remarkable decrease in the PM total lipid amount (Table 1) and can be interpreted as a reduction of the total membrane area of the cells. A lower total lipid content has been found in PSII particles of the same wheat cultivar (Quartacci et al., 2000) and in tomato leaves and roots grown at 50 µM Cu (Ouariti et al., 1997).
Under physiological conditions, FS acts as the main membrane lipid controlling fluidity by increasing the efficiency of PL packing. Free sterols also limit the leakage of electrolytes from roots indicating that they may control membrane permeability as well (Navari-Izzo et al., 1993). The decrease in the FS level of wheat root PM following the 50 µM Cu supply could be responsible, at least in part, for the increased K+ leakage (Fig. 1C; Table 1).
Even though a high PL to FS ratio has been associated with a higher fluidity of the PM (Palta et al., 1993), when an alteration in membrane fluidity is observed it does not necessarily correspond to changes in bulk lipid composition. It has been suggested that there is also a general pattern of increase in the lipid to protein ratio which regulates membrane fluidity (Harwood, 1998; Quartacci et al., 2000). In this study excess Cu lowered the lipid to protein ratio of root PM (Table 1) and, even though direct measurements have not been performed, membrane fluidity might have been decreased as well. It is not fully understood if different steryl lipids exert different effects on membrane fluidity. However, it has been observed that an increase in the proportion of ASG plus FS at the expense of SG could be important in modulating the phase behaviour of PM during stress conditions (Palta et al., 1993; Zhang et al., 1997). The significant decrease of the FS+ASG to SG ratio in the root PM subjected to Cu excess and the altered K+ efflux (Table 1; Fig. 1C) may indicate that changes in sterol conjugation play a role in membrane function (Palta et al., 1993). Excess Cu-induced changes in FS composition were also reflected by the halving of the ratio of ‘more planar’ sterols (cholesterol+campesterol) to ‘less planar’ sterols (sitosterol+stigmasterol). A decrease in this ratio is considered as an index of metabolic disorder and impaired membrane selectivity (Navari-Izzo et al., 1993).
Membrane phase behaviour is also regulated by PL composition and an appropriate balance between bilayer-forming and non-bilayer-forming lipids (Leshem, 1992). Phosphatidylcholine tends to adopt a bilayer configuration, whereas PE becomes oriented into an inverted hexagonal HII configuration (Uemura and Steponkus, 1994). In addition, a high level of choline in the polar head group has a fluidizing effect on the membrane, whereas ethanolamine makes the membrane more rigid (Navari-Izzo et al., 1993; Mansour et al., 1994). The 50 µM Cu supply lowered the PC/PE ratio (Table 2), which may have led to enriched non-lamellar-forming domains and phase separation of non-lamellar-forming lipids (Quinn and Williams, 1984). Furthermore, the concomitant reduction in the FS level may have increased the disorder of the PM lipid bilayer and the tendency of PL demixing and phase separation. Precautions taken during PM isolation and lipid extraction to minimize phospholipase D and other phosphatase and kinase activities resulted in 7% PA in control plants (Table 2). At the present stage of knowledge PA could as well be a minor natural constituent of PM (Moreau et al., 1998). In its turn, the higher PA amount in the PM from roots grown at 50 µM Cu could have affected membrane permeability, due to the segregation of this anionic lipid into clustering or domains (Ahn and Yun, 1998).
The analysis of total PM fatty acids showed a lower unsaturation level in the roots grown without Cu supply and even more in the 50 µM Cu-grown roots (Table 4), which is an indication that a degradative process occurred. Either the direct action of Cu or the hydroxyl radical-mediated mechanism could have played a role in degrading unsaturated acyl chains by the formation of hydroperoxide derivatives, even though the possibility that alterations in PL biosynthesis could be involved in the reduction of the unsaturation levels cannot be excluded. Similar peroxidative damage has been observed in root extracts and thylakoids of plants subjected to Cu exposure (De Vos et al., 1993; Navari-Izzo et al., 1999; Quartacci et al., 2000). Phospholipid fatty acids affect bilayer properties regulating its fluidity and permeability and the activities of membrane-bound enzymes as well (Quartacci et al., 2000). Even though it is unlikely that the fluidity of the bulk lipid phase has any remarkable effect on the function of membrane proteins (Lee et al., 1989), the lower unsaturation of PM specific domains of Cu-treated plants (Table 4) might have contributed to change the lipid environment surrounding integral proteins (Mansour et al., 1994; Quartacci et al., 1995).
Evidence for different types of NAD(P)H-oxidizing activities in PM vesicles from plant tissues, such as those similar to the NADPH oxidase of human neutrophil, is well documented (Van Gestelen et al., 1997). The enzyme(s) catalyses a one electron transfer across the PM to oxygen, producing which in turn originates H2O2. The more harmful and reactive hydroxyl radicals are then produced in a Fenton-type reaction (Halliwell and Gutteridge, 1984), and peroxidative damage to membranes such as degradation of fatty acid double bonds occurs (Table 4). These results show that NADPH-dependent activities of PM-bound oxidases in wheat root cells are affected by the Cu nutritional status of plants (Fig. 2A). An increase in NADPH-dependent -generating activity was already observed in PM, cytosolic and microsomal fractions of zinc-deficient bean roots (Cakmak and Marschner, 1988; Pinton et al., 1994). Furthermore, Fe-deficient roots showed a strongly increased rate of Fe3+ reduction in comparison with Fe-sufficient plants, indicating that a NADPH-dependent enzyme is induced by iron deficiency (Buckhout et al., 1989). As for zinc, the effect of Cu-deficiency on the oxidase activity may be explained by the lack of a specific binding of metals to electron carrying coenzymes which have been seen to inhibit NADPH oxidation and superoxide formation (Ludwig et al., 1980). Thus, it can be suggested that Cu requirement for optimal function and structural integrity of plant membranes is, at least in part, related to the capacity of Cu to interact with PM-bound -generating NADPH oxidases. In addition, as already observed in animal systems, the redox potential of the NADPH-oxidizing complex could be altered by metals, favouring electron transfer to oxygen both in Cu deficiency and excess (Jeffery, 1983) and causing a higher superoxide radical production. It has been hypothesized that Cu itself is directly involved in hydroxyl radical formation (Shinar et al., 1983; Navari-Izzo et al., 1999). In this mechanism free or bound Cu(II) is reduced to Cu(I) by superoxide and react with H2O2 in a Fenton reaction to give the very highly reactive hydroxyl radicals.
It is known that NADPH-derived free radicals are involved in the peroxidation of unsaturated fatty acids in different membrane fractions among which are PM (Cakmak and Marschner, 1988). The slight effect of Cu-deficiency on root cell PM unsaturation (Table 4) can probably be related to a maintained antioxidative defence system. By contrast, when grown at 50 µM Cu wheat cells do not possess an effective scavenging system mechanism against activated oxygen species (Navari-Izzo et al., 1998) and membrane degradation may occur.
Lipoxygenase mediates the conversion of polyunsaturated fatty acids to their conjugated hydroperoxydiene derivatives using molecular oxygen. The maintained or decreased PM-associated LOX activities (Fig. 2B) indicate that no induction of the enzyme occurred following Cu treatments, and that LOX had a minor role in the observed peroxidation of PM fatty acids. Furthermore, the results do not show that additional superoxide radicals were formed as a consequence of LOX activity (Lynch and Thompson, 1984). In evaluating these findings, it is necessary to consider that peroxidative and free radical-generating reactions mediated by LOX are probably determined more by the availability of free fatty acids as a substrate for the enzyme than by changes in its activity (Thompson et al., 1998). Moreover, it cannot be excluded that membrane LOX in vivo is unable to act on phospholipid constituents and thus is not involved in membrane lipid catabolism (Droillard et al., 1993). The lack of LOX activation following Cu deficiency or excess could be due, at least in part, to a non-release of fatty acids from complex lipids. However, it cannot be excluded that the decrease in LOX activity detected at 50 µM Cu supply may be related to a generalized protein degradation which inactivated the enzyme.
Evaluation of the effect of Cu on the cell wall/plasma membrane interface and its mode of action, and studies on the reactive oxygen species and scavenging mechanisms located in the apoplast are in progress.
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Acknowledgments |
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This study was financially supported by the MURST, Rome (Scientific Research Project, ex 40% 1996).
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Notes |
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1 To whom correspondence should be addressed. Fax: +39 050 598614; E-mail: fnavari{at}agr.unipi.it
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Abbreviations |
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PM, plasma membrane; LOX, lipoxygenase; , superoxide;
PL, phospholipids; FS, free sterols; ASG, acylated steryl glycosides; SG, steryl glycosides; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PI, phosphatidylinositol; PS, phosphatidylserine; PA, phosphatidic acid. |
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