Salt and oxidative stress: similar and specific responses and their relation to salt tolerance in Citrus

Salt damage to plants has been attributed to a combination of several factors including mainly osmotic stress and the accumulation of toxic ions. Recent findings in our laboratory showed that phospholipid hydroperoxide glutathione peroxidase (PHGPX), an enzyme active in the cellular antioxidant system, was induced by salt in citrus cells and mainly in roots of plants. Following this observation we studied the two most important enzymes active in elimination of reactive oxygen species, namely, superoxide dismutase (SOD) and ascorbate peroxidase (APX), to determine whether a general oxidative stress is induced by salt. While Cu/Zn-SOD activity and cytosolic APX protein level were similarly induced by salt and methyl viologen, the response of PHGPX and other APX isozymes was either specific to salt or methyl viologen, respectively. Unlike PHGPX, cytosolic APX and Cu/Zn-SOD were not induced by exogenously added abscisic acid. Salt induced a significant increase in SOD activity which was not matched by the subsequent enzyme APX. We suggest that the excess of H₂O₂ interacts with lipids to form hydroperoxides which in turn induce and are removed by PHGPX. Ascorbate peroxidase seems to be a key enzyme in determining salt tolerance in citrus as its constitutive activity in salt-sensitive callus is far below the activity observed in salt-tolerant callus, while the activities of other enzymes involved in the defence against oxidative stress, namely SOD, glutathione reductase and PHGPX, are essentially similar. Received: 10 January 1997 / Accepted: 28 May 1997     

Effects of dietary selenium and vitamin E concentrations on phospholipid hydroperoxide glutathione peroxidase expression in reproductive tissues of pubertal maturing male rats

Phospholipid hydroperoxide glutathione peroxidase (PHGPX) is the second intracellular selenium (Se)-dependent glutathione peroxidase (GSH-Px) identified in mammals. Our objectives were to determine the effect of dietary vitamin E and Se levels on PHGPX activity expression in testis, epididymis, and seminal vesicles of pubertal maturing rats, and the relationship of PHGPX expression with testicular development and sperm quality. Forty Sprague-Dawley male weanling rats (21-d old), were initially fed for 3 wk a torula yeast basal diet (containing 0.05 mg Se/kg) supplemented with marginal levels of Se (0.1 mg/kg as Na₂SeO₃) and vitamin E (25 IU/kg as all-rac-α-tocopheryl acetate). Then, rats were fed the basal diets supplemented with 0 or 0.2 mg Se/kg and 0 or 100 IU vitamin E/kg diet during the 3-wk period of pubertal maturing. Compared with the Se-supplemented rats, those fed the Se-deficient diets retained 31, 88, 67, and 50% of Se-dependent GSH-Px activities in liver, testis, epididymis, and seminal vesicles, respectively. Testes and seminal vesicles had substantially higher (5-to 20-fold) PHGPX activity than liver. Dietary Se deficiency did not affect PHGPX activities in the reproductive tissues, but reduced PHGPX activity in liver by 28% (P < 0.0001). Dietary vitamin E supplementation did not affect PHGPX activity in liver, whereas it raised PHGPX activity in seminal vesicles by 43% (P < 0.005). Neither dietary vitamin E nor Se levels affected body weight gains, reproductive organ weights, or sperm counts and morphology. In conclusion, expression of PHGPX activity in testis and seminal vesicles was high and regulated by dietary Se and vitamin E differently from that in liver.     

苦瓜谷胱甘肽磷脂氢过氧化物酶cDNA的克隆及其特征分析

根据谷胱甘肽磷脂氢过氧化物酶(PHGPX)氨基酸序列中高度保守的区段设计引物,采用RACE-PCR从苦瓜中克隆到一个全长927 bp的cDNA片段.DNA序列的数据库分析比较表明,该cDNA编码167个氨基酸,含有动植物PHGPX的特征结构,是一个新发现的苦瓜PHGPX基因（mocPHGPX）.RNA印迹结果显示,该基因在苦瓜幼苗的根中表达相对较弱,茎的信号较强,叶中最强.这些结果将有助于深入研究植物PHGPX的功能以及全面了解植物抗氧化体系.     

Plant glutathione peroxidases

Oxidative stress in plants causes the induction of several enzymes, including superoxide dismutase (EC 1.15.1.1), ascorbate peroxidase (EC 1.11.1.11) and glutathione reductase (EC 1.6.4.2). The first two are responsible for converting superoxide to H₂O₂ and its subsequent reduction to H₂O, and the third is involved in recycling of ascorbate. Glutathione peroxidases (GPXs, EC 1.11.1.9) are a family of key enzymes involved in scavenging oxyradicals in animals. Only recently, indications for the existence of this enzyme in plants were reported. Genes with significant sequence homology to one member of the animal GPX family, namely phospholipid hydroperoxide glutathione peroxidase (PHGPX), were isolated from several plants. Cit-SAP, the protein product encoded by the citrus csa gene, which is induced by salt-stress, is so far the only plant PHGPX that has been isolated and characterized. This protein differs from the animal PHGPX in its rate of enzymatic activity and in containing a Cys instead of selenocysteine (Sec) as its presumed catalytic residue. The physiological role of Cit-SAP and its homologs in other plants is not yet known.     

Increased Na+ and Cl− accumulation induced by NaCl salinity inhibits cotyledonary reserve mobilization and alters the source-sink relationship in establishing dwarf cashew seedlings

We studied the NaCl-induced changes in cotyledons and the embryonic axis of establishing dwarf cashew (Anacardium occidentale) seedlings. The salt stress reduced the growth of dwarf cashew seedlings, and this response was related to the inhibition of cotyledonary reserve depletion. Lipid mobilization was inhibited by NaCl due to reduced lipase activity in the emerging and establishing seedlings. Additionally, there was reduced transient starch accumulation in the cotyledons of the salt-stressed seedlings that was associated with lower starch synthase activity at the early developmental stages and inhibited amylolytic and starch phosphorylase activities at the established seedling stage. The NaCl-induced changes in lipid and starch metabolism influenced the soluble sugar content in the cotyledons. Protein mobilization was inhibited by NaCl, and we observed the accumulation of amino acids and the inhibition of proteolytic activity in the cotyledons of the salt-stressed established seedlings. Salinity significantly reduced the free amino acid and reducing sugar contents in the embryonic axes of both emerged and established seedlings, whereas the non-reducing sugar content was affected by this stress only in the established seedlings. The Na⁺ and Cl^? contents progressively increased in the cotyledons and embryonic axis of the seedlings as the salinity increased. We conclude that salt stress inhibits dwarf cashew seedling establishment by inhibiting the mobilization of reserves, an inhibition that was related to increased Na⁺ and Cl^? accumulation in the cotyledons. Additionally, these toxic ions reduced the sink strength of the embryonic axis with regard to the products of cotyledonary reserve mobilization.     

Exogenous Melatonin Modulates Endogenous H2S Homeostasis and L-Cysteine Desulfhydrase Activity in Salt-Stressed Tomato (Solanum lycopersicum L. var. cherry) Seedling Cotyledons

Although melatonin has been reported to function as a stress signaling molecule, not much information is available on the biochemical and molecular events associated with probable melatonin-hydrogen sulfide crosstalk in plants. Present work provides evidence on the role of melatonin in the modulation of H₂S homoeostasis during NaCl stress in dark-grown tomato (Solanum lycopersicum L. var. cherry) seedlings. NaCl stress (120 mM) inhibits hypocotyl elongation, promotes primary root growth and enhances electrolytic leakage from tomato seedlings. Treatment with H₂S donor (100 µM; NaHS) tends to reverse these effects, all the more so (additive effect) in the presence of melatonin. NaCl stress and exogenous melatonin (30 µM) treatments modulate endogenous H₂S accumulation and positively upregulate the activity of L-cysteine desulfhydrase (L-DES; EC 4.4.1.15; cytosolic). Melatonin has been observed to temporally modulate the activity of specific isoforms of H₂S biosynthesizing enzyme, L-DES in seedling cotyledons. Zymographic analysis of L-DES isoforms in tomato seedling cotyledons has provided novel findings in plant system. Melatonin treatment decreases H₂S accumulation in NaCl-stressed seedling cotyledons which is accompanied by a contrasting increase in L-DES activity. Melatonin, therefore, regulates endogenous H₂S concentration in seedling cotyledons (NaCl treated), thus indicating the role of H₂S catabolism pathways in H₂S homoeostasis. Present findings thus reveal that exogenous melatonin modulates early H₂S signaling in cotyledons of tomato seedlings subjected to NaCl stress. Furthermore, exogenous melatonin and H₂S in combination (additive effect) ameliorate NaCl stress-induced growth changes in tomato seedlings.

     

Induction of oxidative stress and GPX-like protein activation in tomato plants after mechanical stimulation

In Lycopersicon esculentum Mill. (cv. VFN8), mechanical stimulation induced a rapid and transient increase of with hydrogen peroxide (H₂O₂), a part of an oxidative burst. The reaction was followed by an antioxidative response, with the involvement of phospholipid hydroperoxide glutathione peroxidase (PHGPX)-like protein (EC 1.11.1.9). Induction of expression of two putative PHGPX genes was observed in rubbed internodes. To characterize the importance of this antioxidant gene, enzymatic activities of glutathione peroxidase (GPX) and PHGPX were measured, respectively, H₂O₂ and hydroperoxide lipid as oxidant. Only PHGPX activities were induced by the mechanical treatment, suggesting a major role of PHGPX in the mechanisms of antioxidant defence in plant.     

Role of glutathione peroxidase in protecting mammalian spermatozoa from loss of motility caused by spontaneous lipid peroxidation

Mouse and human spermatozoa, but not rabbit spermatozoa, have long been known to be sensitive to loss of motility induced by exogenous H₂O₂. Recent work has shown that loss of sperm motility in these species correlates with the extent of spontaneous lipid peroxidation. In this study, the effect of H₂O₂ on this reaction in sperm of the three species was investi gated. The rate of spontaneous lipid peroxidation in mouse and human sperm is markedly enhanced in the presence of 1-5 mM H₂O₂, while the rate in rabbit sperm is unaffected by H₂O₂. The enhancement of lipid peroxidation, the rate of reaction of H₂O₂ with the cells, the activity of sperm glutathione peroxidase, and the endogenous glutathione content are highest in mouse sperm, intermediate in human sperm, and very low in rabbit sperm. Inac tivation of glutathione peroxidase occurs in the presence of H₂O₂ due to complete conver sion of endogenous glutathione to GSSG: No GSH is available as electron donor substrate to the peroxidase. Inactivation of glutathione peroxidase by the inhibitor mercaptosucci nate has the same effect on rate of lipid peroxidation and loss of motility in mouse and human sperm as does H₂O₂. This implies that H₂O₂ by itself at 1-5 mM is not intrinsically toxic to the cells. With merceptosuccinate, the endogenous glutathione is present as GSH in mouse and human sperm, indicating that the redox state of intracellular glutathione by itself plays little role in protecting the cell against spontaneous lipid peroxidation. Mouse and human sperm also have high rates of superoxide production. We conclude that the key intermediate in spontaneous lipid peroxidation is lipid hydroperoxide generated by a chain reaction initiated by and utilizing superoxide. Removal of this hydroperoxide by gluta thione peroxidase protects these sperm against peroxidation; inactivation of the peroxidase allows lipid hydroperoxide to increase and so increases the peroxidation rate. Rabbit sperm have low rates of superoxide reaction due to high activity of their superoxide dismutase; lack of endogenous glutathione and low peroxidase activity does not affect their rate or lipid peroxidation. As a result, these sperm are not affected by either H₂O₂ or mercapto-succinate. These results lead us to postulate a mechanism for spontaneous lipid peroxida tion in mammalian sperm which involves reaction of lipid hydroperoxide and O₂ as the rate-determining step.     

Enhancement of stress tolerance in transgenic tobacco plants overexpressing Chlamydomonas glutathione peroxidase in chloroplasts or cytosol

To evaluate the physiological potential of the defense system against hydroperoxidation of membrane-lipid components caused by environmental stresses in higher plants, we generated transgenic tobacco plants expressing a glutathione peroxidase (GPX)-like protein in the cytosol (TcGPX) or chloroplasts (TpGPX). The activities toward alpha-linolenic acid hydroperoxide in TcGPX and TpGPX plants were 47.5-75.3 and 32.7-42.1 nM min(-1) mg(-1) protein, respectively, while no activity was detected in wild-type plants. The transgenic plants showed increased tolerance to oxidative stress caused by application of methylviologen (MV: 50 microM) under moderate light intensity (200 micro E m(-2) sec(-1)), chilling stress under high light intensity (4 degrees C, 1000 microE m(-2) sec(-1)), or salt stress (250 mM NaCl). Under these stresses, the lipid hydroperoxidation (the production of malondialdehyde (MDA)) of the leaves of TcGPX and TpGPX plants was clearly suppressed compared with that of wild-type plants. Furthermore, the capacity of the photosynthetic and antioxidative systems in the transgenic plants remained higher than those of wild-type plants under chilling or salt stress. These results clearly indicate that a high level of GPX-like protein in tobacco plants functions to remove unsaturated fatty acid hydroperoxides generated in cellular membranes under stress conditions, leading to the maintenance of membrane integrity and increased tolerance to oxidative stress caused by various stress conditions.     

The effect of NaCl on antioxidant enzyme activities in potato seedlings

The effect of NaCl on the growth and activity of antioxidant enzymes such as superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) and ascorbate peroxidase (APX) were investigated in the seedlings of four potato cultivars (Agria, Kennebec; relatively salt tolerant, Diamant and Ajax; relatively salt sensitive). The shoot fresh mass of Agria and Kennebec did not changed at 50 mM NaCl, whereas in Diamant and Ajax it decreased to 50 % of that in the controls. In Agria and Kennebec, SOD activity increased at 50 mM NaCl, but no significant changes observed in Diamant and Ajax. At higher NaCl concentration, SOD activity reduced in all cultivars. CAT and POD activities increased in all cultivars under salt stress. Unlike the other cultivars, in Ajax seedlings, APX activity increased in response to NaCl stress. We also observed new POD and SOD isoenzyme activities and changes in isoenzyme compositions under salt stress. These results suggest that salt-tolerant potato cultivars may have a better protection against reactive oxygen species (ROS) by increasing the activity of antioxidant enzymes (especially SOD) under salt stress.     

Antioxidant defense mechanism under salt stress in wheat seedlings

The present study was carried out to study the effect of salt stress on cell membrane damage, ion content and antioxidant enzymes in wheat (Triticum aestivum) seedlings of two cultivars salt-tolerant KRL-19 and salt-sensitive WH-542. Seedlings (4-d-old) were irrigated with 0, 50 and 100 mM NaCl. Observations were recorded on the 3^rd and 6^th day after salt treatment and 2^nd day after salt removal. The relative water content declined with induction of salt stress, more in WH-542 than in cv. KRL-19. K⁺/Na⁺ ratio in KRL-19 was higher than in WH-542. WH-542 suffered greater damage to cellular membranes due to lipid peroxidation as indicated by higher accumulation of H₂O₂, MDA and greater leakage of electrolytes than KRL-19. The activities of catalase, peroxidase and ascorbate peroxidase and glutathione reductase increased with increase in salt stress in both the cultivars, however, superoxide dismutase activity declined. Upon desalanization, partial recovery in the activities of these enzymes was observed in KRL-19 and very slow recovery in WH-542.     

Protective action of phospholipid hydroperoxide glutathione peroxidase against membrane-damaging lipid peroxidation. In situ reduction of phospholipid and cholesterol hydroperoxides

The general reactivity of membrane lipid hydroperoxides (LOOHs) with the selenoenzyme phospholipid hydroperoxide glutathione peroxidase (PHGPX) has been investigated. When human erythrocyte ghosts (lipid content: 60 wt % phospholipid; 25 wt % cholesterol) were treated with GSH/PHGPX subsequent to rose bengal-sensitized photoperoxidation, iodometrically measured LOOHs were totally reduced to alcohols. Similar treatment with the classic glutathione peroxidase (GPX) produced no effect unless the peroxidized membranes were preincubated with phospholipase A2 (PLA2). However, under these conditions, no more than approximately 60% of the LOOH was reduced; introduction of PHGPX brought the reaction to completion. Thin layer chromatographic analyses revealed that the GPX-resistant (but PHGPX-reactive) LOOH was cholesterol hydroperoxide (ChOOH) consisting mainly of the 5 alpha (singlet oxygen-derived) product. Membrane ChOOHs were reduced by GSH/PHGPX to species that comigrated with borohydride reduction products (diols). Sensitive quantitation of PHGPX-catalyzed ChOOH reduction was accomplished by using [14C]cholesterol-labeled ghosts. Kinetic analyses indicated that the rate of ChOOH decay was approximately 1/6 that of phospholipid hydroperoxide decay. Photooxidized ghosts underwent a large burst of free radical-mediated lipid peroxidation when incubation with ascorbate/iron or xanthine/xanthine oxidase/iron. These reactions were only partially inhibited by PLA2/GSH/GPX treatment, but totally inhibited by GSH/PHGPX treatment, consistent with complete elimination of LOOHs in the latter case. These findings provide important clues as to how ChOOHs are detoxified in cells and add new insights into PHGPX's protective role.     

Phospholipid hydroperoxide glutathione peroxidase of rat testis. Gonadotropin dependence and immunocytochemical identification.

A high glutathione peroxidase activity toward phospholipid hydroperoxides is present in rat testis. The attribution of this activity to the selenoenzyme phospholipid hydroperoxide glutathione peroxidase (PHGPX) was supported by cross-reactivity with antibodies raised against pig heart PHGPX which had been purified and characterized. Rat testis PHGPX is partially cytosolic and partially linked to nuclei and mitochondria. The soluble and organelle-bound enzymes appear identical by Western blot analysis. PHGPX, but neither selenium-dependent nor non-selenium-dependent glutathione peroxidase activity, is expressed in testes only after puberty, disappears after hypophysectomy, and is partially restored by gonadotropin treatment. Specific immunostaining of testes by antiserum against PHGPX appears as a fine granular brown pattern localized throughout the cytoplasm in more immature cells but is confined to the peripheral part of the cytoplasm, the nuclear membrane, and mitochondria in maturating spermatogenic cells. As expected, immunostaining of spermatogenic cells in hypophysectomized animals was negative, but gonadotropin treatment only marginally increased the immunoreactivity. The expression of PHGPX in testes is consistent with the previously described specific requirement for selenium for synthesis of a 15-20-kDa selenoprotein which is related to the production of functional spermatozoa.     

Salt stress induces up-regulation of an efficient chloroplast antioxidant system in the salt-tolerant wild tomato species Lycopersicon pennellii but not in the cultivated species

The response of the chloroplastic antioxidant system of the cultivated tomato Lycopersicon esculentum (Lem) and its wild salt-tolerant related species L. pennellii (Lpa) to NaCl stress was studied. An increase in H₂O₂ level and membrane lipid peroxidation was observed in chloroplasts of salt-stressed Lem. In contrast, a decrease in these indicators of oxidative stress characterized chloroplasts of salt-stressed Lpa plants. This differential response of Lem and Lpa to salinity, correlates with the activities of the antioxidative enzymes in their chloroplasts. Increased activities of total superoxide dismutase (SOD), ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), glutathione-S-transferase (GST), phospholipid hydroperoxide glutathione peroxidase (PHGPX) and several isoforms of non-specific peroxidases (POD) were found in chloroplasts of salt-treated Lpa plants. In these chloroplasts, in contrast, activity of lipoxygenase (LOX) decreased while in those of salt-stressed Lem it increased. Although total SOD activity slightly increased in chloroplasts of salt-treated Lem plants, differentiation between SOD types revealed that only stromal Cu/ZnSOD activity increased. In contrast, in chloroplasts of salt-treated Lpa plants FeSOD activity increased while Cu/ZnSOD activity remained unchanged. These data indicate that salt-dependent oxidative stress and damage, suffered by Lem chloroplasts, was effectively alleviated in Lpa chloroplasts by the selective up-regulation of a set of antioxidative enzymes. Further support for the above idea was supplied by leaf discs experiments in which pre-exposure of Lpa plants to salt-treatment conferred cross-tolerance to paraquat-induced oxidative stress while increased oxidative damage by paraquat-treatment was found in salt-stressed Lem plants.     

Effect of NaCl stress on rice physiological properties

Salinity is a major yield-reducing factor in coastal and arid irrigated rice production systems. Rice seedlings (Oryza sativa cv. Tarom Atri) were exposed to different NaCl concentrations for 8 days after germination. Plants height, fresh and dry weight, relative water content, pigment and carbohydrate content, photosynthetic efficiency and lipid peroxidase and antioxidant enzyme activity of rice seedlings grown under salt stress were investigated. Seedling grown under 25and 50 mM salt were shorter than the control. They could, however, develop their secondary leaves. Seedlings grown in the nutrient solution supplied with 100 and 200 mM extra salt could not develop their secondary leaves. Fresh weight ofseedlings grown under salt stress reduced up to 42.2% of the non-treated seedlings. Chlorophylls and carotenoids contents decreased significantly in the salt-treated seedlings. Carotenoid contents in NaCl-treated seedlings were decreased to 39.3%. No significant changes occurred in the photochemical efficiency of control and stressed plants. Increasing concentrations of NaCl resulted in increase and decrease of Na⁺ and K⁺ ions, respectively. NaCl salinity caused an increase in both peroxide content and lipid peroxidation. Seedlings which recovered for 24 h showed lower peroxide and malondialdehyde content.