Behaviour of antioxidant defense system in the adaptive response to salt stress in Helianthus annuus L. cells

A relationship between the antioxidant defense system and salt tolerance in two types of sunflower calli differing in salt sensitivity was studied. No reduction in growth occurred in the NaCl-salt-adapted cell line (T) when grown on 175 mM NaCl but growth of the salt-stressed cell line (S) was reduced by 83%. Lipid peroxidation and protein oxidation increased during acute stress of salt stressed cells at 14 and 28 d of the experiment, while salt-adapted calli (T) remained similar to non-shocked (C) values. The antioxidant defense system of callus adapted to growth under NaCl responded differently to 175 mM of salt compared with the corresponding controls under shock treatment. Salt-adapted and salt-stressed calli showed a similar pattern in GSH content at day 14 but at day 28 in S calli, GSH content was increased 100% over the non-shocked calli, while T calli returned to the initial values. In the salt-stressed calli, a general decrease in all the antioxidant enzymes studied (except for glutathione reductase and dehydroascorbate reductase activities) was observed at day 28. Except for catalase, the antioxidant enzymes were elevated constitutively in adapted calli as compared to stressed cells, when both were grown in the absence of NaCl (time 0), and remained unaltered until 28 d after the beginning of the experiment. These results suggest the involvement of an enzymatic antioxidant defense system in the adaptive response to salt stress in Helianthus annuus L. cells.     

Influence of exogenous proline on embryogenic and organogenic maize callus subjected to salt stress

The effect of exogenous proline (6 mM) and increasing NaCl doses (from 0.4 to 1.2% w/v) on the maintenance of organogenic and embryogenic callus lines derived from the salt-sensitive maize inbred W64Ao2 were studied. To this end, total protein, free amino acid and polyamine content were analyzed. The demand of exogenous nitrogen and especially of proline, even in the presence of salt, differed in the two types of morphogenic calluses. The total protein content of embryogenic calluses was higher in the presence of proline than in its absence, in all the cases studied. An opposite effect of proline was observed in organogenic calluses: the presence of proline and salt decreased significantly their protein content. With respect to amino acid and polyamine contents, the organogenic calluses showed physiological characteristics of salt-adaptation, whereas the embryogenic calluses were more sensitive to NaCl. Although endogenous proline increased in the organogenic calluses cultured in the presence of salt, in embryogenic calluses it only rose at the lowest salt concentration. Furthermore, the endogenous arginine content under saline conditions was higher in organogenic calluses. A compensatory effect between proline and polyamine metabolism related to the endogenous arginine content in response to salt stress was also observed. This effect differed in the two types of calluses.     

Influence of exogenous proline on embryogenic and organogenic maize callus subjected to salt stress

The effect of exogenous proline (6 mM) and increasing NaCl doses (from 0.4 to 1.2% w/v) on the maintenance of organogenic and embryogenic callus lines derived from the salt-sensitive maize inbred W64Ao2 were studied. To this end, total protein, free amino acid and polyamine content were analyzed. The demand of exogenous nitrogen and especially of proline, even in the presence of salt, differed in the two types of morphogenic calluses. The total protein content of embryogenic calluses was higher in the presence of proline than in its absence, in all the cases studied. An opposite effect of proline was observed in organogenic calluses: the presence of proline and salt decreased significantly their protein content. With respect to amino acid and polyamine contents, the organogenic calluses showed physiological characteristics of salt-adaptation, whereas the embryogenic calluses were more sensitive to NaCl. Although endogenous proline increased in the organogenic calluses cultured in the presence of salt, in embryogenic calluses it only rose at the lowest salt concentration. Furthermore, the endogenous arginine content under saline conditions was higher in organogenic calluses. A compensatory effect between proline and polyamine metabolism related to the endogenous arginine content in response to salt stress was also observed. This effect differed in the two types of calluses.     

Production of Polyamines Is Enhanced by Endogenous Abscisic Acid in Maize Seedlings Subjected to Salt Stress

It is known that salt stress and exogenously applied abscisic acid （ABA） can enhance the polyamine content in plants and that salt stress itself can lead to an increase in endogenous ABA production. In the present study, the relationships between salt-induced ABA and polyamine accumulation were inves- tigated using ABA-deficient mutant （vp5/vp5） maize （Zea mays L.） seedlings and ABA and polyamine biosynthesis inhibitors. The results show that reduced endogenous ABA levels, as a result of either the mutation or by using a chemical inhibitor （sodium tungstate）, also reduced the accumulation of polyamines in salt-stressed leaves of maize seedlings. The polyamine synthesis inhibitors D-arginine and α- difluoromethylornithine also reduced the polyamine content of the leaves of maize seedling under salt stress. Both ABA and polyamine enhanced the dry weight accumulation of salt-stressed seedlings and also increased the activities of the two dominant tonoplast membrane enzymes, H^＋-ATPase and H^＋-PPase, when plants were under salt stress. The results suggest that salt stress induces an increase in endogenous ABA levels, which then enhances polyamine synthesis. Such responses may increase a plant＇s tolerance to salt.     

Modulation of polyamine levels in ginseng hairy root cultures subjected to salt stress

Modulation of differential gene expression and change of polyamine content by salt stress are analyzed for the first time in a well-known medicinal plant, Panax ginseng C.A. Meyer. Three ginseng genes (PgSPD, PgSAMDC, and PgADC) involved in polyamine biosynthesis showed differential up-regulation patterns after 1 and 7 days of salt treatments. The modulation of gene expression resulted in the elevation of total polyamine content with relatively high levels of spermidine and spermine, while putrescine level diminished depending on the salt concentration. Conversely, salt stress led to a significant increase in diamine oxidase and subsequent decline in polyamine oxidase. The proline content caused by salinity follows a similar pattern as the total polyamine content and exogenous spermidine also resulted in the alleviation of proline content under salinity. Further, polyamine biosynthesis inhibitors, such as cyclohexylamine and methylglyoxal bis-(guanylhydrazone) mediated down-regulation of PgSPD and PgSAMDC, and affected cellular polyamine levels. Thus, polyamines may enhance the ginseng plant tolerance in response to the salt stress by increasing the levels of endogenous polyamines.     

Modulation of polyamine balance in Lotus glaber by salinity and arbuscular mycorrhiza.

In this work we investigated the involvement of Glomus intraradices in the regulation of plant growth, polyamines and proline levels of two Lotus glaber genotypes differing in salt tolerance, after longterm exposure to saline stress. The experiment consisted of a randomized block design with three factors: (1) mycorrhizal treatments (with or without AM fungus); (2) two salinity levels of 0 and 200mM NaCl; and (3) L. glaber genotype. Experiments were performed using stem cuttings derived from L. glaber individuals representing a natural population from saline lowlands. One of the most relevant results was the higher content of total free polyamines in mycorrhized plants compared to non-AM ones. Since polyamines have been proposed as candidates for the regulation of root development under saline situations, it is possible that AM plants (which contained higher polyamine levels and showed improved root growth) were better shaped to cope with salt stress. Colonization by G. intraradices also increased (Spd+Spm)/Put ratio in L. glaber roots. Interestingly, such increment in salt stressed AM plants of the sensitive genotype, was even higher than that produced by salinization or AM symbiosis separately. On the other hand, salinity but not mycorrhizal colonization influenced proline levels in both L. glaber genotypes since high proline accumulation was observed in both genotypes under salt stress conditions. Our results suggest that modulation of polyamine pools can be one of the mechanisms used by AM fungi to improve L. glaber adaptation to saline soils. Proline accumulation in response to salt stress is a good indicator of stress perception and our results suggest that it could be used as such among L. glaber genotypes differing in salt stress tolerance.     

Effects of salinity on the contents of polyamines and some other compounds in sunflower plants differing in salt tolerance

The effects of salt stress (50, 100, and 150 mM NaCl) on the levels of free, bound, and total polyamines were studied in the leaf tissues of salt-tolerant (Coban) and salt-sensitive (Sanbro) cultivars of sunflower (Helianthus annuus L.) plants grown for 15 or 25 days under salinity. The amounts of free, acid-soluble bound, and total spermine increased in leaf tissues of sunflower plants subjected to salt stress while the levels of other polyamines decreased or no significant changes occurred. The increase in some PA titers suggests their potential role in overcoming the adverse effect of salinity stress. The salt sensitivity of the sunflower plants was associated with the excessive accumulation of total polyamines in the leaf tissues of salt-sensitive cultivar (Sanbro) under saline condition. The content of other compounds such as proline, protein, and ions (Na⁺, K⁺, Cl^-, K⁺/Na⁺) in leaf tissue changed depending on salt concentration and the cultivars used.From Fiziologiya Rastenii, Vol. 52, No. 1, 2005, pp. 36–42.Original English Text Copyright © 2005 by Mutlu, Bozcuk.This article was presented by the authors in English.     

Influence of Ethephon and 2,5-Norbornadiene on Antioxidative Enzymes and Proline Content in Salt-Stressed Spinach Leaves

The effects of ethephon, an ethylene generating compound, and 2,5-norbornadiene (NBD), an inhibitor of ethylene action, on peroxidase (POD; EC 1.11.1.7), catalase (CAT; EC 1.11.1.6), polyphenol oxidase (PPO; EC 1.14.18.1) activities and proline content in salt-stressed spinach leaves were investigated. POD and PPO activities were increased by NaCl + ethephon + NBD combination and reduced by NBD. Also, ethephon increased the CAT activity while ethephon + NBD reduced CAT activity. NaCl + ethephon increased proline content. The antagonistic effect of ethephon and NBD was seen on POD and PPO activity and proline accumulation, but was not on CAT activity.     

Spermine accumulation under salt stress

Polyamines have long been recognized to be linked to stress situations, and it is generally accepted that they have protective characteristics. However, little is known about their physiological relevance in plants subjected to long-term salt stress. In order to precise their importance, two rice (Oryza sativa) cultivars differing in their salt tolerance were salinized for 7, 14 and 21 days. The activities of some of the enzymes involved in polyamine metabolism, free polyamines and proline contents were evaluated. Arginine decarboxylase and S-adenosyl-L-methionine decarboxylase activities were reduced in both cultivars as a consequence of salt treatment. However, spermidine synthase activity was reduced in the salt tolerant cultivar (var Giza) but not in the salt sensitive (var El Paso), while no polyamine oxidase activity was detected. During the salinization period, putrescine and spermidine levels decreased in both cultivars, although less dramatically in Giza. Simultaneously, spermine accumulations occur in both varieties, while proline accumulation was major in the sensitive one. However, spermine accumulation induced by treatment with spermidine synthase inhibitor cyclohexylamine, determined no reduction in leaf injury associated with salt stress in both cultivars. The data presented suggest that spermine accumulation is not a salt tolerance trait.     

Proline accumulation in two bean cultivars under salt stress and the effect of polyamines and ornithine

Proline accumulation in two different bean (Phaseolus vulgaris L.) cultivars, one drought-sensitive (Canario 60) and one drought-resistant (Pinto Villa) was investigated. Both tolerated salt concentrations up to 150 mM NaCl, but the sensitive Canario 60 did not survive at 400 mM NaCl. In response to salt stress, both cvs. accumulated proline in all the analyzed tissues, the lowest contents were detected in roots. Pinto Villa accumulated higher proline concentrations than Canario 60 only at 400 mM NaCl. The addition of polyamines or ornithine increased proline content in plant tissues without stress, while they decreased it under salt stress.     

The effect of salt stress on polyamine biosynthesis and content in mung bean plants and in halophytes

The activity of L-arginine decarboxylase (EC 4.1.1.19) and L-ornithine decarboxylase (EC 4.1.1.17), polyamine content, and incorporation of arginine and ornithine into polyamines, were determined in mung bean [Vigna radiata (L.) Wilczek] plants subjected to salt (hypertonic) stress (NaCl at 0.51–2.27 MPa). Changes in enzyme activity in response to hypotonic stress were determined as well in several halophytes [Pulicaria undulata (L.), Kostei, Salsola rosmarinus (Ehr.) Solms-Laub, Mesembryanthemum forskahlei Hochst, and Atriplex halimus L.]. NaCl stress, possibly combined with other types of stress that accompanied the experimental conditions, resulted in organ-specific changes in polyamine biosynthesis and content in mung bean plants. The activity of both enzymes was inhibited in salt-stressed leaves. In roots, however, NaCl induced a 2 to 8-fold increase in ornithine decarboxylase activity. Promotion of ornithine decarboxylase in roots could be detected already 2 h after exposure of excised roots to NaCl, and iso-osmotic concentrations of NaCl and KCl resulted in similar changes in the activity of both enzymes. Putrescine level in shoots of salt-stressed mung bean plants increased considerably, but its level in roots decreased. The effect of NaCl stress on spermidine content was similar, but generally more moderate, resulting in an increased putrescine/spermidine ratio in salt-stressed plants. Exposure of plants to NaCl resulted also in organ-specific changes in the incorporation of both arginine and ornithine into putrescine: incorporation was inhibited in leaf discs but promoted in excised roots of salt-stressed mung bean plants. In contrast to mung bean (and several other glycophytes), ornithine and arginine decarboxylase activity in roots of halophytes increased when plants were exposed to tap water or grown in a pre-washed soil—i.e. a hypotonic stress with respect to their natural habitat. NaCl, when present in the enzymatic assay mixture, inhibited arginine and ornithine decarboxylase in curde extracts of mung bean roots, but did not affect the activity of enzymes extracted from roots of the halophyte Pulicaria. Although no distinct separation between NaCl stress and osmotic stress could be made in the present study, the data suggest that changes in polyamines in response to NaCl stress in mung bean plants are coordinated at the organ level: activation of biosynthetic enzymes concomitant with increased putrescine biosynthesis from its precursors in the root system, and accumulation of putrescine in leaves of salt-stressed plants. In addition, hypertonic stress applied to glycophytes and hypotonic stress applied to halophytes both resulted in an increase in the activity of polyamine biosynthetic enzymes in roots.     

Effects of NaCl and Proline on Polyphenol Oxidase Activity in Bean Seedlings

In this work, the effects of NaCl (0, 50, 100, and 150 mM), proline (0, 5 and 10 mM) and NaCl + proline in combinations on activity of polyphenol oxidase (PPO; E.C. 1.10.3.1) and soluble protein content have been investigated in the root, stem and leaf tissues of bean (Phaseolus vulgaris L.) seedlings grown in embryo culture. PPO activities were higher in all the tissues treated with NaCl, proline and NaCl + proline combinations those that of the control tissues. The protein content was very high in tissues exposed to proline and NaCl + proline combination, but NaCl alone decreased protein contents in root and leaf tissues. The results suggest that proline may play a role as an enzyme-stabilizing agent in salt stress.     

Polyamine levels in relation to growth and NaCl concentration in normal and habituated sugarbeet callus cultures

Abstract. The concentrations of putrescine, spermidine and spermine, the only polyamines detectable in normal and habituated calli of Beta vulgaris L. ssp. altissima , were much higher in the habituated callus than the normal callus, irrespective of experimental conditions. These results suggest that, in normal (tolerant to NaCl) and habituated (sensitive to NaCl) calli, there exists a competition for the common precursor of ethylene and polyamine biosynthesis viz. S-adenosylmethionine. A disequilibrium favouring the synthesis of putrescine and spermidine in the habituated callus might be linked to structural deterioration of the cell membrane following extended culture or severe osmotic stress (68 mol m⁻¹ NaCl). The maintenance of membrane integrity by the normal callus coincides with ethylene production at the expense of polyamine synthesis. In contrast to the habituated callus, the salinity tolerance of the normal callus is accompanied by the accumulation of proline under hypersaline conditions (274mol m⁻³). The important osmoregulatory role played by quaternary ammonium compounds in the-aerial parts of Chenopodiaceae, especially the sugarbeet, is not observed in the calli, these compounds being found in very low concentrations in saline conditions.     

Salt tolerance in rice in vitro: Implication of accumulation of Na+, K+ and proline

The implication of accumulation of both inorganic (Na⁺, K⁺) and organic (proline) solutes were evaluated in unadapted and NaCl-adapted callus of a salt-sensitive (Basmati 370) and a salt-tolerant (SR-26B) cultivar of rice (Oryza sativa L.) after a NaCl shock. Accumulation of Na⁺,K⁺ and/or proline in callus was co relatable and the relative presence of these components in tissues after shock treatment was found to be important factors to support differential regrowth capacities of the shock treated calluses. Presence or retention of K⁺ in rice callus was a key factor for salt tolerance as it was observed to be positively correlated with growth in both the varieties. The results indicated that K⁺ was the first candidate to counteract the negative water potential of outside milieu, while proline was probably the last metabolic device that rice calluses opted for when exposed to salt stress.     

Variations in endogenous polyamine content of maize calli obtained from zygotic and androgenetic embryos

A comparative study of polyamine (putrescine, spermidine and spermine) levels was conducted with maize calli originating from a) immature embryos and b) pollen embryos capable of plant regeneration. The differences observed in the studied parameters of the two kinds of calluses are related to their cellular origin and to their regeneration capacity. Moreover, only the calluses proceeding from immature embryos differentiated into preembryogenic structures, which eventually developed into plants. Although total polyamine levels in pollenderived calluses were significantly higher than those from immature embryos, spermidine and spermine were the predominant polyamines in both culture types. Furthermore, polyamine fractions of these calluses also showed differences. All these phenomena may be related with the differences observed in the callus embryogenic response. These findings may be useful in understanding the implication of polyaminesin embryogenetic processes.Abbreviations IEC immature-embryo calluses - PAs polyamines - PEC pollen-embryo calluses - PH insoluble conjugated PA fraction - Put putrescine - S free PA fraction - SH soluble conjugated PA fraction - Spd spermidine - Spm spermine 2,4d-2,4 dichlorophenoxyacetic acid     

Biosynthetic Pathways of Glycerol Accumulation under Salt Stress in Aspergillus nidulans

Redkar, R. J., Locy. R. D., and Singh, N. K. 1995. Biosynthetic pathways of glycerol accumulation under salt stress in Aspergillus nidulans. Experimental Mycology 19, 241-246. A culture of Aspergillus nidulans (FGSC 359) was gradually adapted for growth in media containing up to 2 M NaCl or was exposed to a salt shock with 2 M NaCl. The intracellular glycerol level increased by about 7.9-fold in salt-adapted and 2.4-fold in salt-shocked cultures when compared to the unadapted culture. The biosynthetic pathway involved in the accumulation of glycerol was investigated under long-term salt adaptation and short-term salt shock. Glycerol 3-phosphate dehydrogenase (EC 1.1.1.8) was induced 1.4-fold in salt-shocked but not in salt-adapted cultures. An alternate enzymatic pathway involving glycerol dehydrogenase (NADP⁺-dependent) utilizing dihydroxyacetone (DHA) and/or DL-glyceraldehyde (DL-GAD) was induced by NaCl. DHA-dependent glycerol dehydrogenase activity was induced about 6.3-fold in salt adapted and 1.35-fold in salt-shocked cultures, while DL-GAD-dependent activity was induced about 6.1-fold in salt-adapted and 1.2-fold in salt shocked cultures. However, the level of glycerol dehydrogenase activity with DL-GAD as substrate was 7% of the DHA-dependent activity. We conclude that a salt-inducible NADP⁺-dependent glycerol dehydrogenase activity electrophoretically indistinguishable from previously described glycerol dehydrogenase I results in glycerol accumulation in salt-stressed A. nidulans.     

Salicylic acid differently impacts ethylene and polyamine synthesis in the glycophyte Solanum lycopersicum and the wild‐related halophyte Solanum chilense exposed to mild salt stress

This study aimed to determine the effects of exogenous application of salicylic acid (SA) on the toxic effects of salt in relation to ethylene and polyamine synthesis, and to correlate these traits with the expression of genes involved in ethylene and polyamine metabolism in two tomato species differing in their sensitivity to salt stress, Solanum lycopersicum cv Ailsa Craig and its wild salt‐resistant relative Solanum chilense. In S. chilense, treatment with 125 mM NaCl improved plant growth, increased production of ethylene, endogenous salicylic acid and spermine. The production was related to a modification of expression of genes involved in ethylene and polyamine metabolism. In contrast, salinity decreased plant growth in S. lycopersicum without affecting endogenous ethylene, salicylic or polyamine concentrations. Exogenous application of salicylic acid at 0.01 mM enhanced shoot growth in both species and affected ethylene and polyamine production in S. chilense. Concomitant application of NaCl and salicylic acid improved osmotic adjustment, thus suggesting that salt and SA may act in synergy on osmolyte synthesis. However, the beneficial impact of exogenous application of salicylic acid was mitigated by salt stress since NaCl impaired endogenous SA accumulation in the shoot and salicylic acid did not improve plant growth in salt‐treated plants. Our results thus revealed that both species respond differently to salinity and that salicylic acid, ethylene and polyamine metabolisms are involved in salt resistance in S. chilense.     

Metabolism of polyamines in NaCl-resistant cell lines from Nicotiana sylvestris

The polyamine titers in three cell lines of Nicotiana sylvestris were compared: Type 1, rapidly adapting to NaCl; Type 2, constantly resistant to NaCl; Type 3, a saltsensitive wild strain. During short-term cultivation in MS medium in the presence of 170 mM NaCl (1 passage, 14 d) the changes in polyamine titer in cell suspensions of type 1 (in a slightly adapted state) and non-adapted wild strain (type 3) showed a considerable increase in spermidine and spermine and a decrease in putrescine. After prolonged adaptation to NaCl (20 passages) the putrescine content in the cells of type 1 and of type 2 was increased at the expense of the polyamines. This suggests that the pattern of polyamine titer varies under short- and long-term adaptation to NaCl. The inverse ratio between growth processes and changes in polyamine and proline level indicates that polyamines fulfil primarily a protective and osmorepulatory function in plant cells under NaCl stress.     

盐胁迫下大麦幼苗多胺的种类和状态与多胺氧化酶活性的关系

200 mmol/L的NaCl胁迫8 d大麦幼苗叶片和根系中的三种形态多胺都有不同程度地下降,其中游离态多胺含量的下降幅度最大;高氯酸不溶性结合态多胺含量变化较小.根系中PAO的活性先上升后下降,而叶片中PAO的活性先下降后上升.游离态多胺中,亚精胺和精胺(Spd Spm)的含量变化与相应部位PAO的活性变化趋势相反,表明PAO在盐胁迫下可能调节了游离态多胺的含量从而影响高氯酸可溶结合态与高氯酸不溶结合态多胺的含量.