Changes in Lipid Peroxidation and Lipolytic and Free-Radical Scavenging Enzyme Activities during Aging and Sprouting of Potato (Solanum tuberosum) Seed-Tubers

Previous research has shown that cell membranes of potato (Solanum tuberosum L. cv Russet Burbank) seed-tubers lose integrity between 7 and 26 months of storage (4[deg]C, 95% relative humidity), and this loss coincides with a significant decrease in growth potential. The age-induced decline in membrane integrity is apparently due to increased peroxidative damage of membrane lipids. Malondialdehyde (MDA) and ethane concentrations (sensitive markers of lipid peroxidation and membrane damage) increased in seed-tuber tissues with advancing age. Moreover, in vivo ethane production from discs of cortex tissue from 13- and 25-month-old seed-tubers was 87% greater (on average) than that from discs from 1-month-old tubers. Calcium suppressed ethane production from all ages of tissue discs, and the effect was concentration dependent. Linoleic acid enhanced ethane production from 5- and 17-month-old tubers by 61 and 228%, respectively, suggesting that older tissue may contain a higher free-radical (FR) titer and/or lower free polyunsaturated fatty acid content. In addition, throughout plant establishment, the internal ethane concentration of older seed-tubers was 54% higher than that of younger seed-tubers. MDA concentration of tuber tissue declined by about 65% during the initial 7 months of storage and then increased 267% as tuber age advanced to 30 months. The age-induced trend in tuber reducing sugar concentration was similar to that of MDA, and the two were linearly correlated. The age-dependent increase in reducing sugars may thus reflect peroxidative degeneration of the amyloplast membrane, leading to increased starch hydrolysis. Compared with 5-month-old seed tubers, 17- and 29-month-old seed-tubers had significantly higher levels of lipofuscin-like fluorescent compounds (FCs), which are produced when MDA reacts with free amino acids. Age-dependent increases in MDA, ethane, and FCs were not associated with higher activities of phospholipase and lipoxygenase in tissue from older tubers. In fact, 8-month-old seed-tubers had significantly higher activities of these enzymes than 20-month-old seed-tubers. However, the activities of superoxide dismutase, peroxidase, and catalase in 20-month-old tubers were substantially higher out of storage, and increased at a faster rate during plant establishment, than in 8-month-old seed-tubers. Collectively, these results suggest that a gradual build-up of FRs leads to peroxidative damage of membrane lipids during aging of potato seed-tubers.     

Lipid metabolism during aging of high-alpha-linolenate-phenotype potato tubers

Previous studies demonstrated that high levels of alpha-linolenate in cell membranes of potato tubers (achieved by overexpressing fatty acid desaturases) enhances lipid peroxidation, oxidative stress, and tuber metabolic rate, effectively accelerating the physiological age of tubers. This study details the changes in lipid molecular species of microsomal and mitochondrial membranes from wild-type (WT) and high-alpha-linolenate tubers during aging. The microsomal and mitochondrial polar lipids of high-alpha-linolenate tubers were dominated by 18:3/18:3 and 16:0/18:3 molecular species. Relative to WT tubers, high-alpha-linolenate tubers had a substantially higher 16:0/18:n to 18:n/18:n molecular species ratio in mitochondria and microsomes, potentially reflecting a compensatory response to maintain membrane biophysical properties in the face of increased unsaturation. Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) accounted for 53 and 37% of polar lipids, respectively, in mitochondria from younger WT and high-alpha-linolenate tubers. The relative proportions of these phospholipids (PL) did not change during aging of WT tubers. In contrast, PE increased to dominate the PL pool of mitochondria during aging of high-alpha-linolenate tubers. While aging effected an increase in mitochondrial 18:3-bearing PCs and PEs in WT tubers, the concentration of 18:3-bearing PCs fell with a concomitant increase in 18:3-bearing PEs during aging of high-alpha-linolenate tubers. These age- and high-alpha-linolenate-induced changes had no effect on the respiration rate and functional integrity of isolated mitochondria. Differential increases in the respiration rates of WT and high-alpha-linolenate tubers during aging were therefore a consequence of unsaturation-dependent alterations in the microenvironments of cells. Microsomal 18:3-bearing PCs, PEs, digalactosyldiacylglycerols (DGDG), and monogalactosyldiacylglycerols all increased in WT tubers during aging. In contrast, a selective loss of 18:3-bearing PCs and DGDGs from microsomes of high-alpha-linolenate tubers likely reflects a greater susceptibility of membranes to peroxidative catabolism during aging. Aging resulted in an increase in sterol/PL ratio in microsomes from WT tubers, due primarily to a decline in PL. In high-alpha-linolenate tubers, the increase in sterol/PL ratio during aging was due to increases in Delta 5-avenasterol and stigmasterol, indicating membrane rigidification and likely contributing to increased membrane permeability. Age-induced changes in 18:3-bearing lipids in membranes of transformed tubers are discussed relative to the development of oxidative stress and accelerated aging.     

The ascorbate system and lipid peroxidation in stored potato (Solanum tuberosum L.) tubers

The changes in the components of the ascorbateglutathione systemduring the storage of potato (Solanum tuberosum L. cv. Spunta)tubers for 40 weeks at both 3C and 9C were studied in relationto lipid peroxidation. The level of malondialdehyde was foundto be higher at 3C than at 9C throughout storage. Thus, lipidperoxidation, which is the main cause of membrane deterioration,was accelerated at the lower temperature. Catalase activityincreased throughout storage independently of temperature. Theascorbate content of tubers decreased during storage both at3C and at 9C, as in other ageing processes. However, ascorbateperoxidase activity reached a maximum after about 8 weeks ofstorage, then declined at 9C, but held a higher level at 3C.The dehydroascorbic content also reached a maximum after about8 weeks and was significantly higher in tubers stored at 3C.These findings indicate a greater utilization of ascorbate byascorbate peroxidase at the lower temperature. Ascorbate freeradical reductase, dehydroascorbate reductase and glutathionereductase, the enzymes involved in the regeneration of ascorbate,were not affected by temperature and remained quite unchangedthroughout storage. It can be concluded that the ascorbate systemis involved in the scavenging of the free-radicals responsiblefor lipid peroxidation in stored potato tubers, at least atlow temperatures and in the first period of storage. Key words: Ascorbate, lipid peroxidation, potato tubers, Solanum tuberosum L     

Assessing potato tuber diel growth by means of X‐ray computed tomography

The formation and development of belowground organs is difficult to study. X‐ray computed tomography (CT) provides the possibility to analyse and interpret subtle volumetric changes of belowground organs such as tubers, storage roots and nodules. Here, we report on the establishment of a method based on a voxel dimension of 240 μm and precision (standard deviation) of 30 μL that allows interpreting growth differences among potato tubers happening within 3 h. Plants were not stressed by the application of X‐ray radiation, which was shown both by morphological comparison with control plants and by analysis of lipid peroxidation as a measure of oxidative stress. Diel (24 h) tuber growth fluctuations of three potato genotypes were monitored in soil‐filled pots of 10 L. In contrast to the results from previous reports, most tubers grew at similar rates during day and night. Tuber growth was not related to the developmental stage of plants and tubers. Pronounced differences were observed between average growth rates in different tubers within a plant. These results are discussed in the context of restrictions of past methods to study tuber growth and in the context of their potential for the characterization of the formation and development of other belowground plant organs.     

An unconventional hexacoordinated flavohemoglobin from Mycobacterium tuberculosis

Being an obligate aerobe, Mycobacterium tuberculosis faces a number of energetic challenges when it encounters hypoxia and environmental stress during intracellular infection. Consequently, it has evolved innovative strategies to cope with these unfavorable conditions. Here, we report a novel flavohemoglobin (MtbFHb) from M. tuberculosis that exhibits unique features within its heme and reductase domains distinct from conventional FHbs, including the absence of the characteristic hydrogen bonding interactions within the proximal heme pocket and mutations in the FAD and NADH binding regions of the reductase domain. In contrast to conventional FHbs, it has a hexacoordinate low-spin heme with a proximal histidine ligand lacking imidazolate character and a distal heme pocket with a relatively low electrostatic potential. Additionally, MtbFHb carries a new FAD binding site in its reductase domain similar to that of D-lactate dehydrogenase (D-LDH). When overexpressed in Escherichia coli or Mycobacterium smegmatis, MtbFHb remained associated with the cell membrane and exhibited D-lactate:phenazine methosulfate reductase activity and oxidized D-lactate into pyruvate by converting the heme iron from Fe(3+) to Fe(2+) in a FAD-dependent manner, indicating electron transfer from D-lactate to the heme via FAD cofactor. Under oxidative stress, MtbFHb-expressing cells exhibited growth advantage with reduced levels of lipid peroxidation. Given the fact that D-lactate is a byproduct of lipid peroxidation and that M. tuberculosis lacks the gene encoding D-LDH, we propose that the novel D-lactate metabolizing activity of MtbFHb uniquely equips M. tuberculosis to balance the stress level by protecting the cell membrane from oxidative damage via cycling between the Fe(3+)/Fe(2+) redox states.     

Polyamine metabolism of potato seed-tubers during long-term storage and early sprout development

Growth potential of potato (Solanum tuberosum L.) plants is influenced by seed-tuber age. After 24 days of growth, single-eye seedcores from 7-month-old seed-tubers produced 64% more foliar dry matter than those from 19-month-old seed-tubers, reflecting a higher growth rate. This study was initiated to determine if differences in polyamine (PA) metabolism are associated with aging and age-reduced vigor of potato seed-tubers. As tubers aged in storage, putrescine (Put) increased 2.2-fold, while spermidine (Spd) and spermine (Spm) decreased 33% and 38%, respectively. Ethylene content of the tuber tissue also increased with advancing age, suggesting that during the aging process S-adenosylmethionine was directed toward ethylene biosynthesis at the expense of the PAs. Single-eye cores from 7- and 19-month-old tubers were sown and PA levels in core and shoot tissues were monitored during plant development. Put titer of younger cores increased 8.8-fold by 12 days. In contrast, the increase in Put over the initial titer in older cores was 2.9-fold. The reduced ability of older cores to synthesize Put during plant establishment is probably due to a 45% decline in ornithine decarboxylase activity between 12 and 16 days after planting. Lack of available Put substrate limited the biosynthesis of Spd and Spm, and thus their concentrations remained lower in older cores than in younger cores. Lower PA titer in older cores during plant establishment is thus coincident with reduced growth potential. Concentrations of Put and Spd were higher in shoots developing from older cores throughout the study, but there was no age-related difference in Spm content. In contrast, activities of arginine and S-adenosylmethionine decarboxylases were higher in shoots from younger cores during establishment. The results indicate that aging affects PA metabolism in both tuber and developing plant tissues, and this may relate to loss of growth potential with advancing seed-tuber age.     

Age of potato seed-tubers influences protein synthesis during sprouting

The effect of seed-tuber age on the ability of tuber tissue to synthesize protein during sprouting was examined. As seed-tuber age advanced from 4 to 32 months (at 4°C, 95% relative humidity), soluble protein concentration of tubers decreased linearly, with a concomitant increase in free amino acid concentration. The age-induced loss of tuber protein may thus be due to increased proteolysis, decreased protein synthesis, or both. Five- and 17-month-old seed-tubers were compared for their ability to incorporate radiolabeled amino acids into soluble protein at equivalent stages of sprout development. Tuber respiration was profiled through each sprouting stage to characterize the physiological status of the seed-tubers prior to incorporation studies. Five-month-old seed-tubers maintained a constant rate of respiration during sprouting. In contrast, respiration of 17-month-old tubers increased as sprout dry matter increased, resulting in a 2- to 3-fold greater respiratory rate from the older tubers, relative to the younger tubers, at similar stages of sprout development. Prior to sprouting, the rate of incorporation of amino acids into trichloroacetic acid-precipitable protein of tissue from 5-month-old tubers was 2. 9-fold higher than that from 17-month-old tubers. More importantly, protein-synthetic capacity of tissue from younger tubers increased about 1. 7-fold during sprout development. Despite the higher respiratory activity and faster total sprout dry matter accumulation from older seed-tubers, protein synthesis remained at a low and constant level through all stages of sprouting. Protein-synthetic capacity thus declines with advancing tuber age, and this may contribute to reduced growth potential during the latter stages of establishment by affecting the ability of seed-tubers to synthesize enzymes involved in mobilization and translocation of tuber reserves to developing plants.     

Comparison between Ageing of Slices and Ethylene or Rindite Treatments of Whole Potato Tubers: Studies on Microsomal and Mitochondria1 Enzymatic Activities

When potato tubers (Solanum tuberosum L.) are treated with Rindite(ethylene chlorohydrin, ethylene dichloride, carbon tetrachloride,7/3/1, v/v/v) for 2 d, and then stored at 20°C, importantchanges in enzymatic activities are observed in mitochondrialas well as in microsomal fractions isolated from these tubers.In mitochondria isolated from Rindite-treated tubers, the oxidationofsuccinate was more active and more resistant to cyanide thanin mitochondria from untreated tubers. The functioning of acyanide-resistant, SHAM-sensitive oxidative activity was similarto that observed after an ageing treatment of tuber slices oran ethylene treatment of whole tubers. However, in the lattercase, changes in oxidative properties or cyanide-resistancewere less important. Moreover, all the observed changes disappearedafter 4 d of storage. The NADH-femcyanide reductase and NADHcytochromec reductase activities of microsomal fractions isolated fromRindite-treated tubers increased after 1 d or 4 d of storage,relative to the activities of untreated tubers. Moreover, theoleatedesaturase activity, which could not be detccted in microsomesprepared from untreated tubers, was high in microsomes fromtreated tubers stored for 1 d and 4 d, and then the activitydecreased. The induction of an oleatedesaturase activity byRindite treatment can be compared to the effect of the ageingtreatment on tuber slices. The enhancement of oleatedesaturaseactivity was linked to an increase in cytochrome b5content ofmicrosomes of treated tubers. However, all these changes werenot observed when whole tubers were treated with ethylene alone. Key words: Rindite, Mitochondria, Microsomes, Solanum tuberosum     

Membrane lipid dynamics and lipid peroxidation in the early stages of low‐temperature sweetening in tubers of Solanum tuberosum

Sugar accumulation and membrane lipid parameters associated with membrane permeability in chilling injury and senescence were followed in the early stages of low‐temperature sweetening in Solanum tuberosum tubers to monitor their dynamics. Norchip, a low‐temperature sweetening‐susceptible potato cultivar, and North Dakota 860‐2, a low‐temperature sweetening‐tolerant selection, were stored for 55 days at 4 and 12°C. Sugar accumulations were not linear and were characterized by fluctuations or cycles over storage time. Sucrose cycling and accumulation were greatest for Norchip tubers stored at 4°C as compared to the other treatments. Increases in membrane permeability were not detected by increases in electrolyte leakage. No significant changes in the phospholipid, galactolipid, free sterol levels or phospholipid to free sterol ratio were observed. The double bond index obtained from the fatty acid profiles of the total lipid fraction decreased significantly (decreased unsaturation) for Norchip tubers at 4°C over time. Free fatty acid and diene conjugation values fluctuated and increased over time for all treatments with greater amplitude of fluctuations observed for Norchip tubers stored at 4°C. These latter effects may be due to the high levels of lipid acyl hydrolase and lipoxygenase found in potato tubers. When free fatty acid and diene conjugation values were plotted with glucose accumulation over time, a possible relationship among the variables was revealed. The observed peroxidation products could relate low‐temperature stress and the resultant low‐temperature sweetening to chilling injury and drought stress. The anti‐oxidative potential of potato tubers should be considered for future cultivar development as a mechanism to lessen the severity or rate of low‐temperature sweetening development.     

Analysis of age-associated changes in mitochondrial free radical generation by rat testis

Throughout spermatogenesis, mitochondria undergo a morphological and functional differentiation. Mitochondria are involved in the production of reactive oxygen species (ROS), considered one of the mediators of ageing. Particularly, lipid peroxidation is regarded as a major phenomenon by which ROS can impair cellular function. In the present study, we examined the production of superoxide anion, superoxide dismutase activity and the effect of Fe²⁺/ascorbate induced-lipid peroxidation on the respiratory chain activities of testis mitochondria throughout the process of spermatogenesis and ageing. Mitochondria from rat testes generated superoxide anion, mainly using NADH as substrate, which increased according to age. The activity of SOD is age-dependent and greatly stimulated during the first wave of spermatogenesis, but decreases in adulthood and old age. TBARS concentration was also markedly increased by ageing. The activity of mitochondrial respiratory chain complexes is differentially affected by oxidative stress induced by iron/ascorbate, succinate-dehydrogenase activity being less vulnerable than that of NADH-dehydrogenase and cytochrome c oxidase. The data suggest that ageing is accompanied by reduced activity of SOD, leading to excessive oxidative stress and enhanced lipid peroxidation that compromises the functionality of the electron transport chain. The data support the concept that mitochondrial function is an important determinant in ageing.     

Potato plants lacking the CDSP32 plastidic thioredoxin exhibit overoxidation of the BAS1 2-cysteine peroxiredoxin and increased lipid Peroxidation in thylakoids under photooxidative stress

The CDSP32 protein (chloroplastic drought-induced stress protein of 32 kD) is a thioredoxin participating in the defense against oxidative damage. We recently have identified in vitro the BAS1 2-Cys peroxiredoxin, a peroxide-detoxifying enzyme, as a target for CDSP32. Here, we report the characterization under stress conditions of transgenic potato (Solanum tuberosum) plants lacking CDSP32 with regard to the BAS1 redox state and the level of lipid peroxidation. Under control conditions, BAS1 is present at similar levels both in wild-type (WT) and transgenic plants. Under drought and methyl viologen treatment, CDSP32-lacking plants display, compared with WT, an increased proportion of BAS1 monomer corresponding to an overoxidized form of the protein. Leaf discs from transgenic plants treated with methyl viologen exhibit earlier degradation of BAS1 than WT plants do. Using several approaches, i.e. a probe emitting fluorescence when reacting with peroxides, high-performance liquid chromatography determination of lipid hydroxy fatty acid content, and measurement of chlorophyll thermoluminescence, we show a higher lipid peroxidation level under methyl viologen treatment in thylakoids from CDSP32-lacking plants compared with WT. These data show that CDSP32 is a critical component in the defense system against lipid peroxidation in photosynthetic membranes, likely as a physiological electron donor to the BAS1 peroxiredoxin.     

Age-related changes in xanthine oxidase activity and lipid peroxidation,as well as in the correlation between both parameters,in plasma and several organs from female mice

Xanthine oxidase, a purine catabolism enzyme, has been implicated as an important source of oxidant production and plays an essential role in several inflammatory and oxidative stress-related diseases. It is known that the increasing levels of oxidants cause the chronic oxidative stress characteristic of the ageing process. The aim of the present work was to determine the changes in xanthine oxidase activity and oxidative damage to lipids in several organs (liver, kidney, spleen, lung and two different brain areas, namely cerebral cortex and brainstem) and plasma from two different age groups of BALB/c female mice: adult (7-month-old) and old (18-month-old) mice, as well as to analyse the possible correlation between both parameters. Xanthine oxidase activity was significantly increased in liver, cerebral cortex and plasma from old mice in comparison with adults. Similar results were obtained in the lipid peroxidation levels, in which old mice showed a high increment in liver and cerebral cortex. Moreover, the results show a significant and positive correlation between xanthine oxidase activity and lipid peroxidation levels in cerebral cortex. The age-related increase in the xanthine oxidase activity and lipid peroxidation in liver and cerebral cortex of mice seems to suggest that the xanthine oxidase plays a role in the acceleration of the oxidative damage in these organs with age and its possible contribution to the pathophysiological changes associated to the process of ageing.     

Quercetin 3-glucoside protects neuroblastoma (SH-SY5Y) cells in vitro against oxidative damage by inducing sterol regulatory element-binding protein-2-mediated cholesterol biosynthesis

The flavonoid quercetin 3-glucoside (Q3G) protected SH-SY5Y, HEK293, and MCF-7 cells against hydrogen peroxide-induced oxidative stress. cDNA microarray studies suggested that Q3G-pretreated cells subjected to oxidative stress up-regulate the expression of genes associated with lipid and cholesterol biosynthesis. Q3G pretreatment elevated both the expression and activation of sterol regulatory element-binding protein-2 (SREBP-2) only in SH-SY5Y cells subjected to oxidative stress. Inhibition of SREBP-2 expression by small interfering RNA or small molecule inhibitors of 2,3-oxidosqualene:lanosterol cyclase or HMG-CoA reductase blocked Q3G-mediated cytoprotection in SH-SY5Y cells. By contrast, Q3G did not protect either HEK293 or MCF-7 cells via this signaling pathway. Moreover, the addition of isopentenyl pyrophosphate rescued SH-SY5Y cells from the inhibitory effect of HMG-CoA reductase inhibition. Last, Q3G pretreatment enhanced the incorporation of [(14)C]acetate into [(14)C]cholesterol in SH-SY5Y cells under oxidative stress. Taken together, these studies suggest a novel mechanism for flavonoid-induced cytoprotection in SH-SY5Y cells involving SREBP-2-mediated sterol synthesis that decreases lipid peroxidation by maintaining membrane integrity in the presence of oxidative stress.     

Cadmium-induced changes in antioxidative systems,hydrogen peroxide content,and lipid peroxidation in<Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

To study the relationship between cadmium (Cd)-induced phytotoxicity and oxidative stress, we grew Cd-sensitive wild-type (WT) and Cd-resistant type (RT) seedlings ofArabidopsis thaliana on MS media containing up to 500 μM CdCl₂. The resistant seedlings showed higher biomasses and lower hydrogen peroxide and lipid peroxidation levels, the latter expressed in terms of malondialdehyde (MDA) production. These results indicate that RT plants experience lower oxidative stress when exposed to Cd. Furthermore, compared with the WT, RT seedlings have significantly higher activities of superoxide dismutase (SOD) and enzymes related to hydrogen peroxide removal, e.g., guaiacol peroxidase (GPX), ascorbate peroxidase (APX), and glutathione reductase (GR). These differential responses suggest that such phytotoxicity could be induced by oxidative stress, and that lower accumulations of hydrogen peroxide confer Cd tolerance in seedlings.     

Lipoxygenase Pathway and Membrane Permeability and Composition during Storage of Potato Tubers (Solanum tuberosum L. cv Bintje and Désirée) in Different Conditions

Abstract: Potato tubers ( Solanum tuberosum L. cv Bintje and Désirée) were stored for 12 months under three different storage conditions: 4 °C, 20 °C with sprout inhibitor and 20 °C without sprout inhibitor. Independent of the storage conditions, our results show that the increase of membrane permeability, as revealed by electrolyte leakage, is not correlated with the lipid saturation status. Moreover, there is no simple correlation between cold sweetening and membrane permeability or lipid saturation status. During storage at 20 °C without sprout inhibitor, the increase in membrane permeability is inversely correlated to sucrose accumulation, but this is not the case when tubers were stored with sprout inhibitors. Lipoxygenase (LOX) is often proposed as responsible for peroxidative damage to membrane lipids. The gradual peroxidation resulting in double bond index decrease is regarded as a cause of senescence sweetening. Our results revealed that the role of LOX in aging and senescence of potato tubers is far from clear. LOX activity and gene expression are not correlated with the fatty acids composition of the membrane. Moreover, LOX activity and fatty acid hydroperoxide content are low in older tubers, whatever the storage conditions or the varieties. On the basis of our results, the correlation between sugar accumulation (low temperature and senescence sweetening) and peroxidative damage occurring during storage of potato tubers is discussed.     

Mitochondrial respiratory chain dysfunction in ageing; influence of vitamin E deficiency

The causes and consequences of ageing are likely to be complex and involve the interaction of many processes. It has been proposed that the decline in mitochondrial function caused by the accumulation of oxidatively damaged molecules plays a significant role in the ageing process. In agreement with previous reports we have shown that the activities of NADH CoQ₁ reductase and cytochrome oxidase declined with increasing age in both rat liver and gastrocnemius muscle mitochondria. However, only in the liver were the changes in lipid peroxidation and membrane fluidity suggestive of an age-related increase in oxidative stress.

After 12 weeks on a vitamin E deficient diet, vitamin E levels were undetectable in both gastrocnemius muscle and liver. In skeletal muscle, this was associated with a statistically significant increase in lipid peroxidation, a decrease in cytochrome oxidase activity after 48 weeks, and an exacerbation in the age-related rate of decline of NADH CoQ₁ reductase activity. This was consistent with the suggestion that an imbalance between free radical generation and antioxidant defence may contribute to the mitochondrial dysfunction with age. In contrast to this, vitamin E deficiency in the liver caused a significant increase in mitochondrial respiratory chain activities with increasing age despite evidence of increased lipid peroxidation. Comparison of other features in these samples suggested vitamin E deficiency; did not have a significant impact upon mtDNA translation; induced a compensatory increase in glutathione levels in muscle, which was less marked in the liver, but probably most interestingly caused a significant decrease in the mitochondrial membrane fluidity in muscle but not in liver mitochondria.

These data suggest that while increased lipid peroxidation exacerbated the age-related decline in muscle respiratory chain function this relationship was not observed in liver. Consequently other factors are likely to be contributing to the age-related decline in mitochondrial function and specific stimuli may influence or even reverse these age-related effects as observed with vitamin E deficiency in the liver.     

Effects of 24-epibrassinolide on seed germination, seedling growth, lipid peroxidation, proline content and antioxidative system of rice (Oryza sativa L.) under salinity stress

The effects of 24-epibrassinolide (24-epiBL) on seedling growth, antioxidative system, lipid peroxidation, proline and soluble protein content were investigated in seedlings of the salt-sensitive rice cultivar IR-28. Seedling growth of rice plants was improved by 24-epiBL treatment under salt stress conditions. When seedlings treated with 24-epiBL were subjected to 120 mM NaCl stress, the activities of superoxide dismutase (EC 1.15.1.1), catalase (EC 1.11.1.6) and glutathione reductase (EC 1.6.4.2) did not show significant difference, whereas the activity of ascorbate peroxidase (EC 1.11.1.11) significantly increased. Increased activity of peroxidase (EC 1.11.1.7) under NaCl stress showed remarkable decrease in the 24-epiBL+NaCl-applied group. Lipid peroxidation level significantly increased under salt stress but decreased with 24-epiBL application revealing that less oxidative damage occurred in this group (24-epiBL+NaCl). In addition, increased proline content in the NaCl-applied group was decreased by 24-epiBL application in the 24-epiBL+NaCl-applied group. Soluble protein content was increased by 24-epiBL application even under NaCl stress, being also higher than control conditions (no 24-epiBL or NaCl treatment). 24-epiBL treatment considerably alleviated oxidative damage that occurred under NaCl-stressed conditions and improved seedling growth in part under salt stress in sensitive IR-28 seedlings.     

Overexpression of Loquat Dehydrin Gene <Emphasis Type="Italic">EjDHN1</Emphasis> Promotes Cold Tolerance in Transgenic Tobacco

Dehydrins (DHNs) play vital roles in response to dehydration stress in plants. To examine the contribution of EjDHN to low-temperature stress in loquat (Eriobotrya japonica Lindl.), EjDHN1 was overexpressed in tobacco (Nicotiana tabacum L.). The plant growth of transgenic lines was significantly better than wild type (WT) after 4 d of recovery from cold stress. Cold stress led to membrane lipid peroxidation and reduced photosystem II (PSII) activity in leaves, and these were less severe in transgenic lines. To examine oxidative stress tolerance, the plants were treated with different concentrations of methyl viologen (MV), which inhibited plant growth both in WT and transgenic lines. After exposure to 2.0 μM MV for 10 d, the WT plants had a dramatically lower survival rate. MV treatment in leaf disks confirmed that transgenic lines accumulated less reactive oxygen species (ROS) and suffered less lipid peroxidation. The results suggested that the tolerance of the transgenic plants to cold was increased, and EjDHN1 could protect cells against oxidative damage caused by ROS production under cold stress. It also provided evidences that the enhanced cold tolerance resulted from EjDHN1 overexpression could be partly due to their protective effect on membranes by alleviating oxidative stresses.     

Functional significance of the pentose phosphate pathway and glutathione reductase in the antioxidant defenses of human sperm

Glutathione peroxidase is one of the principal antioxidant defense enzymes in human spermatozoa, but it requires oxidized glutathione to be reduced by glutathione reductase using NADPH generated in the pentose phosphate pathway. We investigated whether flux through the pentose phosphate pathway would increase in response to oxidative stress and whether glutathione reductase was required to protect sperm from oxidative damage. Isotopic measurements of the pentose phosphate pathway and glycolytic flux, thiobarbituric acid assay of malondialdehyde for lipid peroxidation, and computer-assisted sperm analysis for sperm motility were assessed in a group of normal, healthy semen donors. Applying moderate oxidative stress to human spermatozoa by adding cumene hydroperoxide, H(2)O(2), or xanthine plus xanthine oxidase or by promoting lipid peroxidation with ascorbate increased flux through the pentose phosphate pathway without changing the glycolytic rate. However, adding higher concentrations of oxidants inhibited both the pentose phosphate pathway and glycolytic flux. At concentrations of 50 microg/ml or greater, the glutathione reductase-inhibitor 1,3-bis-(2-chloroethyl) 1-nitrosourea decreased flux through the pentose phosphate pathway and blocked the response to cumene hydroperoxide. It also increased lipid peroxidation and impaired the survival of motility in sperm incubated under 95% O(2). These data show that the pentose phosphate pathway in human spermatozoa can respond dynamically to oxidative stress and that inhibiting glutathione reductase impairs the ability of sperm to resist lipid peroxidation. We conclude that the glutathione peroxidase-glutathione reductase-pentose phosphate pathway system is functional and provides an effective antioxidant defense in normal human spermatozoa.     

Regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase and lipid metabolism in a concanavalin A-resistant Chinese hamster ovary cell line

Lipid metabolism in a concanavalin A-resistant, glycosylation-defective mutant cell line was investigated by comparing growth properties, lipid composition, and lipid biosynthesis in wild-type (WT), mutant (CR-7), and revertant (RCR-7) cells. In contrast to WT and RCR-7, the mutant was auxotrophic for cholesterol, but mevalonolactone did not restore growth on lipoprotein-deficient medium. The use of R-[2-14C]mevalonolactone revealed that CR-7 was deficient in the conversion of lanosterol to cholesterol. Total lipid and phospholipid content and composition were similar in all three cell lines, but CR-7 displayed subnormal content and biosynthesis of cholesterol and unsaturated fatty acids. The mutant was hypersensitive to compactin and was unable to upregulate either 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity or the binding and internalization of 125I-labeled low-density lipoprotein (LDL) in response to lipoprotein deprivation. HMG-CoA reductase activity in all three cell lines showed similar kinetics and phosphorylation status, and the binding kinetics and degradation of 125I-LDL were also similar, suggesting that CR-7 possesses kinetically normal reductase and LDL binding sites, but is deficient in their coordinate regulation. Tunicamycin (1-2 micrograms/ml) strongly and reversibly suppressed reductase activity in WT and RCR-7. CR-7 was resistant to this inhibitor. In WT cells this suppressive effect was accompanied by inhibition of 3H-labeled mannose incorporation into cellular protein, but 3H-labeled leucine incorporation was unaffected. Immunotitration of HMG-CoA reductase activity in extracts of WT cells, cultured in the presence and absence of tunicamycin, showed that suppression of reductase activity reflected the presence of reduced amounts of reductase protein, implying that glycosylation plays an important role in the coordinate regulation of HMG-CoA reductase activity and LDL binding.