Role of lipid composition and lipid peroxidation in the sensitivity of fungal plant pathogens to aluminum chloride and sodium metabisulfite

Aluminum chloride and sodium metabisulfite have shown high efficacy at low doses in controlling postharvest pathogens on potato tubers. Direct effects of these two salts included the loss of cell membrane integrity in exposed pathogens. In this work, four fungal potato pathogens were studied in order to elucidate the role of membrane lipids and lipid peroxidation in the relative sensitivity of microorganisms exposed to these salts. Inhibition of mycelial growth in these fungi varied considerably and revealed sensitivity groups within the tested fungi. Analysis of fatty acids in these fungi demonstrated that sensitivity was related to high intrinsic fatty acid unsaturation. When exposed to the antifungal salts, sensitive fungi demonstrated a loss of fatty acid unsaturation, which was accompanied by an elevation in malondialdehyde content (a biochemical marker of lipid peroxidation). Our data suggest that aluminum chloride and sodium metabisulfite could induce lipid peroxidation in sensitive fungi, which may promote the ensuing loss of integrity in the plasma membrane. This direct effect on fungal membranes may contribute, at least in part, to the observed antimicrobial effects of these two salts.     

Ultrastructural alterations of Erwinia carotovora subsp. atroseptica caused by treatment with aluminum chloride and sodium metabisulfite

Aluminum and bisulfite salts inhibit the growth of several fungi and bacteria, and their application effectively controls potato soft rot caused by Erwinia carotovora. In an effort to understand their inhibitory action, ultrastructural changes in Erwinia carotovora subsp. atroseptica after exposure (0 to 20 min) to different concentrations (0.05, 0.1, and 0.2 M) of these salts were examined by using transmission electron microscopy. Plasma membrane integrity was evaluated by using the SYTOX Green fluorochrome that penetrates only cells with altered membranes. Bacteria exposed to all aluminum chloride concentrations, especially 0.2 M, exhibited loosening of the cell walls, cell wall rupture, cytoplasmic aggregation, and an absence of extracellular vesicles. Sodium metabisulfite caused mainly a retraction of plasma membrane and cellular voids which were more pronounced with increasing concentration. Bacterial mortality was closely associated with SYTOX stain absorption when bacteria were exposed to either a high concentration (0.2 M) of aluminum chloride or prolonged exposure (20 min) to 0.05 M aluminum chloride or to a pH of 2.5. Bacteria exposed to lower concentrations of aluminum chloride (0.05 and 0.1 M) for 10 min or less, or to metabisulfite at all concentrations, did not exhibit significant stain absorption, suggesting that no membrane damage occurred or it was too weak to allow the penetration of the stain into the cell. While mortality caused by aluminum chloride involves membrane damage and subsequent cytoplasmic aggregation, sulfite exerts its effect intracellularly; it is transported across the membrane by free diffusion of molecular SO2 with little damage to the cellular membrane.     

Ultrastructural Alterations of Erwinia carotovora subsp. atroseptica Caused by Treatment with Aluminum Chloride and Sodium Metabisulfite

Aluminum and bisulfite salts inhibit the growth of several fungi and bacteria, and their application effectively controls potato soft rot caused by Erwinia carotovora. In an effort to understand their inhibitory action, ultrastructural changes in Erwinia carotovora subsp. atroseptica after exposure (0 to 20 min) to different concentrations (0.05, 0.1, and 0.2 M) of these salts were examined by using transmission electron microscopy. Plasma membrane integrity was evaluated by using the SYTOX Green fluorochrome that penetrates only cells with altered membranes. Bacteria exposed to all aluminum chloride concentrations, especially 0.2 M, exhibited loosening of the cell walls, cell wall rupture, cytoplasmic aggregation, and an absence of extracellular vesicles. Sodium metabisulfite caused mainly a retraction of plasma membrane and cellular voids which were more pronounced with increasing concentration. Bacterial mortality was closely associated with SYTOX stain absorption when bacteria were exposed to either a high concentration (0.2 M) of aluminum chloride or prolonged exposure (20 min) to 0.05 M aluminum chloride or to a pH of 2.5. Bacteria exposed to lower concentrations of aluminum chloride (0.05 and 0.1 M) for 10 min or less, or to metabisulfite at all concentrations, did not exhibit significant stain absorption, suggesting that no membrane damage occurred or it was too weak to allow the penetration of the stain into the cell. While mortality caused by aluminum chloride involves membrane damage and subsequent cytoplasmic aggregation, sulfite exerts its effect intracellularly; it is transported across the membrane by free diffusion of molecular SO₂ with little damage to the cellular membrane.     

Specificity and mode of action of the antifungal fatty acid cis-9-heptadecenoic acid produced by Pseudozyma flocculosa

cis-9-Heptadecenoic acid (CHDA), an antifungal fatty acid produced by the biocontrol agent Pseudozyma flocculosa, was studied for its effects on growth and/or spore germination in fungi. Inhibition of growth and/or germination varied considerably and revealed CHDA sensitivity groups within tested fungi. Analysis of lipid composition in these fungi demonstrated that sensitivity was related primarily to a low intrinsic sterol content and that a high level of unsaturation of phospholipid fatty acids was not as involved as hypothesized previously. Our data indicate that CHDA does not act directly with membrane sterols, nor is it utilized or otherwise modified in fungi. A structural mechanism of CHDA, consistent with the other related antifungal fatty acids produced by P. flocculosa, is proposed in light of its activity and specificity. The probable molecular events implicated in the sensitivity of fungi to CHDA are (i) partitioning of CHDA into fungal membranes; (ii) a variable elevation in fluidity dependent on the buffering capability (sterol content) in fungi; and (iii) higher membrane disorder causing conformational changes in membrane proteins, increased membrane permeability and, eventually, cytoplasmic disintegration.     

Induction of lipid peroxidation during heavy metal stress in Saccharomyces cerevisiae and influence of plasma membrane fatty acid unsaturation.

The degree of plasma membrane fatty acid unsaturation and the copper sensitivity of Saccharomyces cerevisiae are closely correlated. Our objective was to determine whether these effects could be accounted for by differential metal induction of lipid peroxidation. S. cerevisiae S150-2B was enriched with the polyunsaturated fatty acids (PUFAs) linoleate (18:2) and linolenate (18:3) by growth in 18:2- or 18:3-supplemented medium. Potassium efflux and colony count data indicated that sensitivity to both copper (redox active) and cadmium (redox inactive) was increased in 18:2-supplemented cells and particularly in 18:3-supplemented cells. Copper- and cadmium-induced lipid peroxidation was rapid and associated with a decline in plasma membrane lipid order, detected by fluorescence depolarization measurements with the membrane probe trimethylammonium diphenylhexatriene. Levels of thiobarbituric acid-reactive substances (lipid peroxidation products) were up to twofold higher in 18:2-supplemented cells than in unsupplemented cells following metal addition, although this difference was reduced with prolonged incubation up to 3 h. Conjugated-diene levels in metal-exposed cells also increased with both the concentration of copper or cadmium and the degree of cellular fatty acid unsaturation; maximal levels were evident in 18:3-supplemented cells. The results demonstrate heavy metal-induced lipid peroxidation in a microorganism for the first time and indicate that the metal sensitivity of PUFA-enriched S. cerevisiae may be attributable to elevated levels of lipid peroxidation in these cells.     

Specificity and Mode of Action of the Antifungal Fatty Acid cis-9-Heptadecenoic Acid Produced by Pseudozyma flocculosa

cis-9-Heptadecenoic acid (CHDA), an antifungal fatty acid produced by the biocontrol agent Pseudozyma flocculosa, was studied for its effects on growth and/or spore germination in fungi. Inhibition of growth and/or germination varied considerably and revealed CHDA sensitivity groups within tested fungi. Analysis of lipid composition in these fungi demonstrated that sensitivity was related primarily to a low intrinsic sterol content and that a high level of unsaturation of phospholipid fatty acids was not as involved as hypothesized previously. Our data indicate that CHDA does not act directly with membrane sterols, nor is it utilized or otherwise modified in fungi. A structural mechanism of CHDA, consistent with the other related antifungal fatty acids produced by P. flocculosa, is proposed in light of its activity and specificity. The probable molecular events implicated in the sensitivity of fungi to CHDA are (i) partitioning of CHDA into fungal membranes; (ii) a variable elevation in fluidity dependent on the buffering capability (sterol content) in fungi; and (iii) higher membrane disorder causing conformational changes in membrane proteins, increased membrane permeability and, eventually, cytoplasmic disintegration.     

Lipid peroxidation in rat uterus

Lipid peroxidation in rat uterus has been studied using NADPH- and ascorbate-induced systems. Lipid peroxidation in rat uterus is low as compared to rat liver. Uterus is more sensitive to ascorbate-induced lipid peroxidation than that induced by NADPH. Uterus contains lower amounts of phospholipids and has a lesser degree of unsaturation in lipids. Co-factor studies show that Fe2+ is more important for ascorbate-induced lipid peroxidation. Endometrium is more sensitive to ascorbate-induced lipid peroxidation than myometrium. It also contains more total lipids and phospholipids besides having a higher degree of unsaturation in the lipids as compared to myometrium. Among the subcellular fractions, mitochondria are more prone to ascorbate-induced lipid peroxidation, whereas microsomes are more sensitive to NADPH-induced lipid peroxidation. Uteri from old rats (24 months) and pregnant rats are more resistant to lipid peroxidation than those from 3-month-old control rats. Uterus of pregnant rats contains more factors which inhibit lipid peroxidation and also has a lesser degree of unsaturation in lipids compared with uterus of control rats. The possible consequences of the resistance of uterus to lipid peroxidation, especially during pregnancy and senescence, are discussed.     

脂质过氧化对人红细胞膜脂流动性的影响

研究枯稀过氧化氢/高铁血红素体系所产生的烷基过氧自由基对红细胞的损伤。测定了脂质过氧化的产物——丙二脂的生成,并证明阿魏酸钠对脂质过氧化的抑制。荧光偏振的结果指出,膜脂过氧化以后降低了膜脂的流动性。人红细胞用5DSA和16DSA标记并用ESR检测膜脂流动性,结果表明,序参数S几乎没有发生变化,旋转相关时间τ值的增加证明膜脂过氧化以后,疏水尾部的物理状态发生了改变。经脂质过氧化以后,红细胞膜中的不饱和脂防酸的减少,可能是降低膜脂流动性的原因之一。     

Fatty acids, membrane permeability, and sugars of stored potato tubers

The relationships of potato (Solanum tuberosum L.) tuber membrane permeability and membrane lipid composition to sugar accumulation were examined. Tubers from four potato cultivars were stored for 40 weeks at 3°C and 9°C. Rates of tuber membrane electrolyte leakage, total fatty acid composition, free fatty acid composition, and sugar content were measured throughout the storage period. Storage of tubers at 3°C caused dramatic increases in total fatty acid unsaturation, membrane permeability, and sugar content compared to tubers stored at 9°C. Cultivars with higher levels of fatty acid unsaturation had lower rates of membrane electrolyte leakage and lower sugar contents. We propose that high initial levels or high induced levels of membrane lipid unsaturation mitigate increases in tuber membrane permeability during storage, thus positively influencing the processing quality of stored potato tubers.     

Differences in the susceptibility of plant membrane lipids to peroxidation

Peroxidation of three membrane lipid preparations from plants was initiated using Fe-EDTA and ascorbate and quantified as the production of aldehydes and loss of esterified fatty acids. Using liposomes prepared from commercial soybean asolecithin, the degree of peroxidation was shown to be dependent on: the free radical dose, which was varied by the ascorbate concentration; the presence of tocopherol in the liposome; the configuration, of the liposome, multilamellar or unilamellar; and time after initiation. There were dramatic interactions among these factors which led to the conclusion that in comparing the susceptibility of different membrane preparations it is essential to examine the kinetics of the peroxidation reactions. The composition of the liposome was a major determinant of the degree of peroxidation and of the type of degradative reactions initiated by the oxygen free radicals. A fresh polar lipid extract from Typha pollen had very similar fatty acid composition to the soybean asolecithin, but was more resistant to peroxidation as shown by less aldehyde production and increased retention of unsaturated fatty acids after treatment. Similarly, microsomal membranes from the crowns of non-acclimated and cold acclimated winter wheat (Triticum aestivum L.) seedlings had a much higher linolenic acid content than soybean asolecithin but was much more resistant to peroxidation. In the winter wheat microsomes, the loss of esterified fatty acids was not selective for the unsaturated fatty acids; consequently, even with 40% degradation, the degree of unsaturation in the membrane did not decrease. These different reaction mechanisms which occur in plant membranes may explain why measurements of fatty acid unsaturation fail to detect peroxidative reactions during processes such as senescence, aging and environmental stress.     

Lipid peroxidation in the fungus Curvularia lunata exposed to nickel

The effect of Ni²⁺ on fungal growth, cellular fatty acid profile and lipid peroxidation was studied (with an emphasis on the kinetics of these processes) in the strain of filamentous fungus Curvularia lunata. In the cultures supplemented with 0.2 and 0.6 mM Ni²⁺ the lag phase was extended and the specific growth rate decreased, however, the maximum yield of biomass at the stationary phase reached, respectively, 97 and 27% of the control. The treatment with Ni²⁺ changed the proportion of 18 C atom fatty acids, with the most significant decrease in the content of linoleic acid (18:2) followed by a rise in the degree of fatty acid saturation. In the mycelia exposed to Ni²⁺ the levels of TBARS (lipid peroxidation products) increased and ranged between 156 and 823% over the control. The presented data reveal that the oxidative stress resulting, among others, in membrane lipid peroxidation is involved in the mechanisms of the nickel toxicity towards C. lunata and suggest that this fungus exhibits an ability to cope, to some extent, with the increased level of lipid peroxides.     

A tale of two lipids: Lipid unsaturation commands ferroptosis sensitivity

Membrane lipids play important roles in the regulation of cell fate, including the execution of ferroptosis. Ferroptosis is a non-apoptotic cell death mechanism defined by iron-dependent membrane lipid peroxidation. Phospholipids containing polyunsaturated fatty acids (PUFAs) are highly vulnerable to peroxidation and are essential for ferroptosis execution. By contrast, the incorporation of less oxidizable monounsaturated fatty acids (MUFAs) in membrane phospholipids protects cells from ferroptosis. The enzymes and pathways that govern PUFA and MUFA metabolism therefore play a critical role in determining cellular sensitivity to ferroptosis. Here, we review three lipid metabolic processes—fatty acid biosynthesis, ether lipid biosynthesis, and phospholipid remodeling—that can govern ferroptosis sensitivity by regulating the balance of PUFAs and MUFAs in membrane phospholipids.     

Oxidative metabolism and the physiological age of seed potatoes are affected by increased alpha-linolenate content

The effects of high alpha-linolenate content on lipid peroxidation, oxidative stress and loss of plant growth potential during ageing of potato (Solanum tuberosum L.) seed-tubers was examined. Endoplasmic reticulum (FAD3) and plastidal (FAD7) 18:2 fatty acid desaturases were upregulated in potato (cv. Desiree), resulting in a 2-fold average increase in mol percentage 18:3 in the total lipid fraction across all transgenic clones. In double-transformed (FAD3+7) tubers, high alpha-linolenate phenotype effected accelerated ageing, resulting in growth responses characteristic of older seed-tubers. Although respiration rates of wild-type (WT) and FAD3+7 tubers were equal at 7 months of storage, rates had increased by 23% and 50% in WT and FAD3+7 tubers, respectively, by 19 months of storage. Electrolyte leakage of tissue from 19-month-old FAD3+7 tubers was significantly greater than that from WT tubers of the same age, indicating that the high alpha-linolenate phenotype was detrimental to membrane integrity during long-term storage. On average, indices of lipid peroxidation (malondialdehyde, ethane, C-6 aldehydes) were higher in older FAD3+7 tubers, relative to WT tubers. Activities of glucose-6-phosphate dehydrogenase, peroxidase, glutathione reductase, ascorbate peroxidase and monodehydroascorbate reductase increased in tubers with advancing age and were higher, on average, in FAD3+7 tubers. Dehydroascorbate reductase activity decreased with age, with no difference between transgenic and WT lines. Collectively, these results indicate that FAD3+7 tubers underwent a higher degree of oxidative stress during ageing. The age-induced increase in respiration of FAD3+7 tubers was at least partly a response to fuel increased free radical scavenging through the ascorbate-glutathione antioxidant pathway. By affecting the susceptibility of lipids to peroxidation, the degree of fatty acid unsaturation influenced the development of oxidative stress and the overall rate at which growth potential was lost from seed-tubers during ageing. Thus, oxidative stress plays an integral role in modulating the ageing process to affect growth potential from potato seed-tubers.     

Heightened susceptibility of fish oil polyunsaturate-enriched neoplastic cells to ethane generation during lipid peroxidation

We have studied the generation of volatile hydrocarbons by fatty acid-modified L1210 leukemia cells in tissue culture as a measure of lipid peroxidation. There was considerable generation of ethane, and this was dependent on cell number and Fe2+ concentration; it was eliminated by antioxidants and augmented by ascorbic acid. The assay was sensitive and reproducible; ethane was detected when as little as 0.03% of the cellular n-3 (omega-3) fatty acids were peroxidized. To gain further understanding we used a lipid modification model that allows study of cells enriched with fatty acids of different degrees of unsaturation. The quantity of ethane generated was greatest by cells modified with fatty acids of the n-3 family, and there was a high direct correlation of percentage of n-3 fatty acids contained in cellular lipids with peroxidation as measured by ethane generation. Ethane generation was more sensitive in detecting peroxidation than loss of polyunsaturated fatty acids. We conclude that lipid-supplemented leukemic cells produce ethane, and that the rate of generation is a sensitive, quantitative, and highly useful measure of lipid peroxidation when small amounts of iron are present.     

Effects of Cadmium on Polar Lipid Composition and Unsaturation in Maize （Zea mays） in Hydroponic Culture

This study aimed to evaluate the effect of Cd exposure （100 μmol/L） on polar lipid composition, and to examine the level of fatty acid unsaturation in maize （Zea mays L.）. In roots, the level of 16：0 and monounsaturated fatty acids （16：1 ＋ 18：1） decreased in phosphatidylcholine （PC） and phosphatidylethanolamine （PE）. In contrast, the proportion of unsaturated 18-C fatty acid species showed an opposite response to Cd. The content, on the other hand, of PC, PE, digalactosyldiacylglycerol （DGDG）, and steryl lipids increased in roots （2.9-, 1.6-, 5.3-, and 1.7-fold increase, respectively）. These results suggest that a more unsaturated fatty acid composition than found in control plants with a concomitant increase in polar lipids may favor seedling growth during Cd exposure. However, the observed increase in the steryl lipid （SL） ： phospholipid （PL） ratio （twofold）, the decrease in monogalactosyldiacylglycerol （MGDG） ： DGDG ratio, as well as the induction of lipid peroxidation in roots may represent symptoms of membrane injury. In shoots, the unsaturation level was markedly decreased in PC and phosphatidylglycerol （PG） after Cd exposure, but showed a significant increase in sulfoquinovosyldiacylglycerol （SQDG）, MGDG and DGDG. The content of PG and MGDG was decreased by about 65%, while PC accumulated to higher levels （4.4-fold increase）. Taken together, these changes in the polar lipid unsaturation and composition are likely to be due to alterations in the glycerolipid pathway. These results also support the idea that the increase in overall unsaturation plays some role in enabling the plant to withstand the metal exposure.     

Effects of Methylprednisolone and the Combination of α-Tocopherol and Selenium on Arachidonic Acid Metabolism and Lipid Peroxidation in Traumatized Spinal Cord Tissue

Traumatic injury of the spinal cord leads to a series of pathological events that result in tissue necrosis and paralysis. Among the earliest biochemical reactions are hydrolysis of fatty acids from membrane phospholipids, production of biologically active eicosanoids, and peroxidation of lipids. This study examines the effect of agents purported to improve recovery following spinal cord trauma, methylprednisolone sodium succinate (MPSS) and the combination of alpha-tocopherol and selenium (Se), on the posttraumatic alterations of membrane lipid metabolism. Pretreatment with either MPSS or alpha-tocopherol and Se reduced the trauma-induced release of total FFA including arachidonate in the injured spinal cord tissue. In addition, these agents decreased the postinjury levels of prostanoids. Pretreatment with either MPSS or alpha-tocopherol and Se also completely prevented the trauma-induced loss of cholesterol while inhibiting the increase of a cholesterol peroxidation product, 25-hydroxycholesterol. These data suggest that: perturbation of membrane lipid metabolism may contribute to the tissue necrosis and functional deficit of spinal cord injury and MPSS or the combination of alpha-tocopherol and Se may protect injured spinal cord tissue, at least in part, by limiting these posttraumatic membrane lipid changes.     

极长链脂肪酸的合成缺陷对酵母细胞膜的稳定性和多烯类药物敏感性的影响

【背景】脂肪酸延长酶家族参与脂肪酸代谢具有真核生物的高度保守性,且与膜脂的代谢密切相关。但细胞极长链脂肪酸(Very long-chain fatty acid,VLCFA)的合成缺陷对膜的稳定性及多烯类药物的敏感性影响并不完全明晰。【目的】探究细胞VLCFA延长酶ELO1、ELO2和ELO3的作用及功能。【方法】研究脂肪酸延长酶缺陷型elo1?、elo2?和elo3?对多烯类药物两性霉素B (Amphotericin B,AmB)、制霉菌素(Nystatin,Ny)及唑类硝酸益康唑(Econazolenitrate,Eco)的响应,检测不同酵母细胞的麦角固醇,检测其对Na+的响应及胞内钠钾离子水平。【结果】发现细胞VLCFA延长酶ELO2和ELO3缺陷后对AmB高度敏感;VLCFA延长酶缺陷突变株elo2?和elo3?对其它多烯类药物Ny及唑类药物Eco也十分敏感;细胞膜不饱和脂肪酸增加也会改变膜的稳定性,实验结果表明外源油酸(Oleic acid,OLA)增加了elo2?和elo3?突变体的AmB敏感性;相对野生型BY4741和elo1?,缺陷菌株elo2?和elo3?中麦角固醇的含量有显著下降;钠钾离子平衡是维护细胞正常生理的必要条件,也是检测细胞膜稳定性的重要参数,发现VLCFA的合成缺陷菌株对高浓度的NaCl比野生型菌株更敏感,使用ICP-AES检测不同浓度AmB胁迫下细胞内钠钾离子水平,也显示VLCFA延长酶缺陷菌株中,钠水平表现出上升趋势,并且细胞内钾含量明显降低。【结论】细胞VLCFA的合成缺陷会导致细胞膜更脆弱、稳定性下降,从而提高真菌对多烯类药物的敏感性,也表明脂肪酸延长酶是潜在的抗真菌治疗靶点。     

渗透调节对冷敏感大豆[Glycine max(L.)Merr.]种子膜类脂组成和脂肪酸含量的影响

选用冷敏感的大豆[Gzycine max（L.）Merr．]品种“中黄22”为试材,运用薄层层析法和气相色谱法研究了渗控增强大豆种子活力过程中膜类脂的组成及变化规律,揭示生物膜类脂在抗吸胀冷害中的作用。结果表明,用33％（W／V）的PEG6000处理种子,随着渗控时间的延长,种子的发芽指数、活力指数和根干重都逐渐增加,在渗控72h时,大豆种子中不饱和磷脂如PC、PE的比例增加,各极性脂的脂肪酸组成也发生了变化,PC、PE和PI中饱和脂肪酸16：0的含量显著下降,而不饱和脂肪酸18：2明显上升,使它们的不饱和指数大大提高,并与渗控时间呈正相关。由以上结果可证明,经PEG渗控处理后,提高了冷敏感大豆膜的流动性,为防止吸胀冷害,保持膜系统的完整性和生物膜正常的代谢提供了前提条件。     

Lipid peroxidation is an early symptom triggered by aluminum, but not the primary cause of elongation inhibition in pea roots

Pea (Pisum sativum) roots were treated with aluminum in a calcium solution, and lipid peroxidation was investigated histochemically and biochemically, as well as other events caused by aluminum exposure. Histochemical stainings were observed to distribute similarly on the entire surface of the root apex for three events (aluminum accumulation, lipid peroxidation, and callose production), but the loss of plasma membrane integrity (detected by Evans blue uptake) was localized exclusively at the periphery of the cracks on the surface of root apex. The enhancement of four events (aluminum accumulation, lipid peroxidation, callose production, and root elongation inhibition) displayed similar aluminum dose dependencies and occurred by 4 h. The loss of membrane integrity, however, was enhanced at lower aluminum concentrations and after longer aluminum exposure (8 h). The addition of butylated hydroxyanisole (a lipophilic antioxidant) during aluminum treatment completely prevented lipid peroxidation and callose production by 40%, but did not prevent or slow the other events. Thus lipid peroxidation is a relatively early symptom induced by the accumulation of aluminum and appears to cause, in part, callose production, but not the root elongation inhibition; by comparison, the loss of plasma membrane integrity is a relatively late symptom caused by cracks in the root due to the inhibition of root elongation.     

Lipid peroxidation initiated by superoxide-dependent hydroxyl radicals using complexed iron and hydrogen peroxide

Iron salts stimulate lipid peroxidation by decomposing lipid peroxides to produce alkoxyl and peroxyl radicals which initiate further oxidation. In aqueous solution ferrous salts produce OH. radicals, a reactive species able to abstract hydrogen atoms from unsaturated fatty acids, and so can initiate lipid peroxidation. When iron salts are added to lipids, containing variable amounts of lipid peroxide, the former reaction is favoured and OH. radicals contribute little to the observed rate of peroxidation. When iron is complexed with EDTA, however, lipid peroxide decomposition is prevented, but the complex reacts with hydrogen peroxide to form OH. radicals which are seen to initiate lipid peroxidation. Superoxide radicals appear to play an important part in reducing the iron complex.