Over-expression of the apple <Emphasis Type="Italic">spermidine synthase</Emphasis> gene in pear confers multiple abiotic stress tolerance by altering polyamine titers

An apple spermidine synthase (SPDS) gene (MdSPDS1) was verified to encode a functional protein by the complementation of the spe3 yeast mutant, which lacks the SPDS gene. To justify our hypothesis that apple SPDS is involved in abiotic stress responses and to obtain transgenic fruit trees tolerant to abiotic stresses as well, MdSPDS1-over-expressing transgenic European pear (Pyrus communis L. ‘Ballad’) plants were created by Agrobacterium-mediated transformation. A total of 21 transgenic lines showing various spermidine (Spd) titers and MdSPDS1 expression levels were obtained. Selected lines were exposed to salt (150 mM NaCl), osmosis (300 mM mannitol), and heavy metal (500 μM CuSO₄) stresses for evaluating their stress tolerances. Transgenic line no. 32, which was revealed to have the highest Spd accumulation and expression level of MdSPDS1, showed the strongest tolerance to these stresses. When growth increments, electrolyte leakage (EL), and values of thiobarbituric acid reactive substances (TBARS) were monitored, line no. 32 showed the lowest growth inhibition and the least increase in EL or TBARS under stress conditions. Spd titers in wild-type and transgenic lines showed diverse changes upon stresses, and these changes were not consistent with the changes in MdSPDS1 expressions. Moreover, there were no differences in the sodium concentration in the shoots between the wild type and line no. 32, whereas the copper concentration was higher in the wild type than in line no. 32. Although the mechanism(s) underlying the involvement of polyamines in stress responses is not known, these results suggest that the over-expression of the SPDS gene substantially increased the tolerance to multiple stresses by altering the polyamine titers in pear. Thus, MdSPDS1-over-expressing transgenic pear plants could be used to improve desert land and/or to repair polluted environments. Xiao-Peng Wen and Xiao-Ming Pang contributed equally to this work.     

Aluminum tolerance in a spermidine synthase-overexpressing transgenic European pear is correlated with the enhanced level of spermidine via alleviating oxidative status

Aluminum (Al) stress is a major cause of poor crop yields, particularly in those countries where acid soil predominate. To verify whether polyamine can confer Al tolerance, in vitro shoots of a transgenic European pear (Pyrus communis L. ‘Ballad’) line #32 overexpressing apple spermidine synthase (MdSPDS1) and the wild type (WT) were subjected to long-term stress for 30 μM AlCl₃. Based on net increment of shoot height (SHI) or fresh weight (FWI) and morphological changes upon the stress, the performance of line #32 was much better than that of WT. Although SPDS expression levels and spermidine (Spd) titers in line #32 were higher than those in WT, firmly due to the transgene (MdSPDS1) expression, no further induction of SPDS expression was observed from the long-term Al stress trial in both lines. While, Spd titers were considerably increased in both lines after the stress. The activities of superoxide dismutase (SOD) or glutathione reductase (GR) and the accumulation of proline or malondialdehyde (MDA) altered upon this stress toward a more favorable status for survival in the transgenic line #32 than in WT. These antioxidant parameters were closely related to Spd titer. Concentrations of calcium (Ca) and some co-factor metals of SOD in line #32 were diversely higher than that in WT after the stress. These evidences indicate that Spd is implicated in elevating of Al stress tolerance of the transgenic line #32 chiefly via ameliorating oxidative status as well as by affecting mineral element balance.     

Aluminum tolerance in a spermidine synthase-overexpressing transgenic European pear is correlated with the enhanced level of spermidine via alleviating oxidative status

Aluminum (Al) stress is a major cause of poor crop yields, particularly in those countries where acid soil predominate. To verify whether polyamine can confer Al tolerance, in vitro shoots of a transgenic European pear (Pyrus communis L. ‘Ballad’) line #32 overexpressing apple spermidine synthase (MdSPDS1) and the wild type (WT) were subjected to long-term stress for 30 μM AlCl₃. Based on net increment of shoot height (SHI) or fresh weight (FWI) and morphological changes upon the stress, the performance of line #32 was much better than that of WT. Although SPDS expression levels and spermidine (Spd) titers in line #32 were higher than those in WT, firmly due to the transgene (MdSPDS1) expression, no further induction of SPDS expression was observed from the long-term Al stress trial in both lines. While, Spd titers were considerably increased in both lines after the stress. The activities of superoxide dismutase (SOD) or glutathione reductase (GR) and the accumulation of proline or malondialdehyde (MDA) altered upon this stress toward a more favorable status for survival in the transgenic line #32 than in WT. These antioxidant parameters were closely related to Spd titer. Concentrations of calcium (Ca) and some co-factor metals of SOD in line #32 were diversely higher than that in WT after the stress. These evidences indicate that Spd is implicated in elevating of Al stress tolerance of the transgenic line #32 chiefly via ameliorating oxidative status as well as by affecting mineral element balance.     

Antisense inhibition of a spermidine synthase gene highlights the role of polyamines for stress alleviation in pear shoots subjected to salinity and cadmium

Three transgenic European pear (Pyrus communis L.) lines with reduced spermidine synthase (SPDS) expression and spermidine (Spd) titers were developed using a construct containing an apple SPDS gene (MdSPDS1) in antisense orientation. After exposure to either salt or cadmium stress, growth inhibition was more severe in the antisense lines than in the wild-type (WT). The antioxidant system, as shown by glutathione (GSH) content, activity of glutathione reductase (GR) and superoxide dismutase (SOD), and proline accumulation, was not effectively induced under stress in the antisense lines as compared with the WT. The reduction in antioxidant system function in the antisense lines was accompanied by a greater accumulation of malondialdehyde (MDA), an indicator of lipid peroxidation. Growth inhibition, Spd level, and parameters indicative of the antioxidant system were significantly ameliorated by exogenous Spd application. Under either salt or cadmium stress, GSH content, GR and SOD activity, and proline accumulation were positively correlated with Spd, putrescine (Put), and total polyamine titers. Conversely, MDA level showed a significantly negative correlation with these polyamines under both stress conditions. Thus, the responses to stress treatments were first identified in the SPDS antisense European pears, and the results provide further evidence for the important role of polyamines in both salt and cadmium stress tolerance, in which the polyamines act, at least in part, by influencing the antioxidant system.     

Spermidine levels are implicated in heavy metal tolerance in a <Emphasis Type="Italic">spermidine synthase</Emphasis> overexpressing transgenic European pear by exerting antioxidant activities

To verify whether spermidine synthase (SPDS) can confer long-term multi-heavy metal tolerance, in vitro shoots of a transgenic European pear (Pyrus communis L. ‘Ballad’) line #32 overexpressing apple SPDS (MdSPDS1), as well as a wild type (WT) line, were subjected to stress using either CdCl₂, PbCl₂, ZnCl₂, or a combination thereof. Based on either shoot height increment or fresh weight and morphological changes upon heavy metal stress, the performance of the transgenic line #32 was better than that of WT. Although SPDS expression levels and spermidine (Spd) contents in line #32 were higher than those in WT, possibly due to transgene (MdSPDS1) expression, no obvious inductions of SPDS expression and increases in Spd-content were observed by long-term stress treatments in both lines. When the glutathione (GSH) content was compared with or without stress in each line, GSH was significantly depleted in line #32 with stress, but not as much as in WT. The activities of glutathione reductase and superoxide dismutase and the content of malondialdehyde, an indicator for lipid peroxidation, changed upon stress toward a more favorable status for survival in line #32 than in WT. These antioxidant parameters were positively related to Spd-content. The accumulation of heavy metals tended to be less in line #32 than in WT except for Zn stress, and the Ca content showed an opposite trend. These results suggest that Spd-levels are implicated in enhanced heavy metal tolerance, possibly by exerting an antioxidant activity as well as by the properties of Spd per se including metal chelator.     

Effects of exogenous spermidine on antioxidant system of tomato seedlings exposed to high temperature stress

The effects of foliar spraying with spermidine (Spd) on antioxidant system in tomato (Lycopersicon esculentum Mill.) seedlings were investigated under high temperature stress. The high temperature stress significantly inhibited plant growth and reduced chlorophyll (Chl) content. Application of exogenous 1 mM Spd alleviated the inhibition of growth induced by the high temperature stress. Malondialdehyde (MDA), hydrogen peroxide (H₂O₂) content and superoxide anion (O₂) generation rate were significantly increased by the high temperature stress, but Spd significantly reduced the accumulation of reactive oxygen species (ROS) and MDA content under the stress. The high temperature stress significantly decreased glutathione (GSH) content and activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), ascorbate peroxidase (APX), glutathione reductase (GR), monodehydroascorbate reductase (MDHAR) and dehydroascorbate reductase (DHAR), but increased contents of dehydroascorbic acid (DHA), ascorbic acid (AsA), and oxidized glutathione (GSSG) in tomato leaves. However, Spd significantly increased the activities of antioxidant enzymes, levels of antioxidants and endogenous polyamines in tomato leaves under the high temperature stress. In addition, to varying degrees, Spd regulated expression of MnSOD, POD, APX2, APX6, GR, MDHAR, DHAR1, and DHAR2 genes in tomato leaves exposed to the high temperature stress. These results suggest that Spd could change endogenous polyamine levels and alleviate the damage by oxidative stress enhancing the non-enzymatic and enzymatic antioxidant system and the related gene expression.     

Evolution of putrescine N-methyltransferase from spermidine synthase demanded alterations in substrate binding

Putrescine N-methyltransferase (PMT) catalyses S-adenosylmethionine (SAM)-dependent methylation of putrescine in tropane alkaloid biosynthesis. PMT presumably evolved from the ubiquitous spermidine synthase (SPDS). SPDS protein structure suggested that only few amino acid exchanges in the active site were necessary to achieve PMT activity. Protein modelling, mutagenesis, and chimeric protein construction were applied to trace back evolution of PMT activity from SPDS. Ten amino acid exchanges in Datura stramonium SPDS dismissed the hypothesis of facile generation of PMT activity in existing SPDS proteins. Chimeric PMT and SPDS enzymes were active and indicated the necessity for a different putrescine binding site when PMT developed.     

Protective role of exogenous spermidine against paraquat toxicity in radish chloroplasts

When radish chloroplasts were pretreated with 1 mM spermidine (Spd) and then exposed to 30 M paraquat (PQ), they improved their tolerance to subsequent PQ-induced oxidative damages. That included the decreases in the contents of chlorophyll, protein, and ascorbate, as well as the increases in malondialdehyde (MDA) and H₂O₂ levels. Analysis of antioxidant enzymes showed that Spd pretreatment effectively prevented the PQ-induced decreases in the total activities of superoxide dismutase (SOD) and ascorbate peroxidase (APX). In contrast, the normally enhanced activities of dehydroascorbate reductase (DHAR) and glutathione reductase (GR) in PQ-treated chloroplasts were reversed by Spd pretreatment In a native gel assay, the Cu/ZnSOD isozyme, which disappeared under the PQ alone treatment, was significantly recovered when tissues were pretreated with Spd. The dominant APX4 isozyme activity, which was preferentially decreased in response to PQ alone treatment, was also strongly reactivated by earlier Spd exposure. Therefore, we suggest that Spd could play a substantial role in protecting the radish chloroplasts from PQ stress. Furthermore, the enhancement of the Cu/ZnSOD and APX4 isozymes by Spd pretreatment seems to be responsible for prevention of the PQ-induced decreases in the total activities of SOD and APX, thereby providing a tolerance to PQ toxicity.     

Cloning, expression, characterisation and three-dimensional structure determination of Caenorhabditis elegans spermidine synthase

The polyamine synthesis enzyme spermidine synthase (SPDS) has been cloned from the model nematode Caenorhabditis elegans. Biochemical characterisation of the recombinantly expressed protein revealed a high degree of similarity to other eukaryotic SPDS with the exception of a low affinity towards the substrate decarboxylated S-adenosylmethionine (Km = 110 microM) and a less pronounced feedback inhibition by the second reaction product 5'-methylthioadenosine (IC50 = 430 microM). The C. elegans protein that carries a nematode-specific insertion of 27 amino acids close to its N-terminus was crystallized, leading to the first X-ray structure of a dimeric eukaryotic SPDS.     

Spermidine application enhances tomato seedling tolerance to salinity-alkalinity stress by modifying chloroplast antioxidant systems

The purpose of this study was to elucidate whether exogenous spermidine (Spd) protection of tomato (Solanum lycopersicum L.) seedlings under salinity-alkalinity stress is associated with antioxidant enzymes in the chloroplast. The effects of exogenous Spd on antioxidant enzyme activity and antioxidant content in the chloroplast were evaluated in seedlings of salt-sensitive ecotype (Zhongza 9) grown in a 75 mM salinity-alkalinity solution, with or without 0.25 mM Spd foliar spraying. Results showed that salinity-alkalinity stress increased MDA content, superoxide anion O₂^?- generation rate, superoxide dismutase (SOD), ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR) activities and ratio of AsA/DHA and reduced contents of ascorbate (AsA), dehydroascorbate (DHA), AsA+DHA, glutathione (GSH), oxidized glutathione (GSSG), GSH+GSSG, dehydroascorbate reductase (DHAR) activity and ratio of GSH/GSSG in chloroplasts. The exogenous Spd application combined with salinity-alkalinity stress decreased the O₂^?- generation rate and MDA content compared to salinity-alkalinity stress alone. The exogenous Spd also increased AsA-GSH cycle components and increased all antioxidant enzyme activities in most cases. Therefore, exogenous Spd alleviates salinity-alkalinity stress damage using antioxidant enzymes and non-enzymatic systems in chloroplasts.     

Amelioration of salinity stress by exogenously applied spermidine or spermine in three varieties of indica rice differing in their level of salt tolerance

We present here the comparative protective potentiality of exogenously applied polyamines (PAs), namely spermidine (Spd) and spermine (Spm), in mitigating NaCl toxicity and inducing short-term salinity tolerance in three indica rice varieties, namely M-1-48 (salt-sensitive), Nonabokra (salt-tolerant) and Gobindobhog (highly sensitive). The retardation in root length or shoot length and toxic Na⁺ accumulation or K⁺ loss, the considerable increment in malondialdehyde/H₂O₂ accumulation or lipoxygenase activity, all of which were particularly noteworthy in M-1-48 and Gobindobhog during salinity stress, was appreciably reduced by co-treatment with Spd or Spm. Both the PAs also inhibited the extent of salt-induced protein carbonylation in all the varieties and enhanced protease activity, especially in Gobindobhog. The prevention of chlorophyll degradation was better with Spd in Nonabokra and Gobindobhog. While the salt-induced increase in anthocyanin or reducing sugar level was further prompted by Spd or Spm in all the varieties, the proline content was elevated by Spd particularly in Gobindobhog. During salinity stress, both the PAs were effective in lowering the putrescine accumulation in M-1-48 and Gobindobhog, and strikingly increasing the Spm level in all the varieties, the highest being in Gobindobhog. In addition, they enhanced the activity of peroxidases and compensated for the decreased catalase activity in all the varieties. Thus the two PAs could recuperate all the three varieties from salt-induced damages to different degrees. The salt injuries, encountered in M-1-48 and Gobindobhog, both of which showed greater susceptibility to salinity stress, were more pronouncedly alleviated and counteracted by the PAs, than the salt-tolerant Nonabokra. The reversal of inhibitory effect of salinity stress was conferred by preventing growth inhibition or various forms of cellular damages, maintaining proper K⁺/Na⁺ balance or triggering the level of osmolytes and activity of antioxidant enzymes. Our communication offers a referenced evidence for an understanding of the mechanism by which higher PAs relieve the damages particularly in salt-sensitive rice varieties.     

Exogenous spermidine and spermine enhance cadmium tolerance of <Emphasis Type="Italic">Potamogeton malaianus</Emphasis>

In order to investigate the effects of spermidine (Spd) and spermine (Spm) on cadmium stress, the content of chlorophyll, hydrogen peroxide (H₂O₂), malondialdehyde (MDA), soluble protein and proline, the rate of O₂^·− generation, and activities of antioxidant enzymes (superoxide dismutase (SOD), peroxidase (POD), ascorbate peroxidase (APX), and glutathione reductase (GR)) in Potamogeton malaianus Miq. were measured. Exogenous application of Spd or Spm significantly enhanced the level of proline, retarded the loss of chlorophyll, enhanced photosynthesis, decreased the rate of O₂^·− generation and H₂O₂ content, and prevented Cd-induced lipid peroxidation. Spd and Spm also effectively maintained the balance of antioxidant enzyme activities under Cd stress; however, GR activity was found to increase only slightly in response to polyamines (PAs). The antioxidant systems, which were modified by PAs, were able to moderate the radical-scavenging system and to lessen in this way the oxidative stress. These results suggest that both Spd and Spm can enhance Cd tolerance of P. malaianus.     

Crystal structure of Plasmodium falciparum spermidine synthase in complex with the substrate decarboxylated S-adenosylmethionine and the potent inhibitors 4MCHA and AdoDATO

Plasmodium falciparum is the causative agent of the most severe type of malaria, a life-threatening disease affecting the lives of over three billion people. Factors like widespread resistance against available drugs and absence of an effective vaccine are seriously compounding control of the malaria parasite. Thus, there is an urgent need for the identification and validation of new drug targets. The enzymes of the polyamine biosynthesis pathway have been suggested as possible targets for the treatment of malaria. One of these enzymes is spermidine synthase (SPDS, putrescine aminopropyltransferase), which catalyzes the transfer of an aminopropyl moiety from decarboxylated S-adenosylmethionine (dcAdoMet) to putrescine, leading to the formation of spermidine and 5'-methylthioadenosine. Here we present the three-dimensional structure of P. falciparum spermidine synthase (pfSPDS) in apo form, in complex with dcAdoMet and two inhibitors, S-adenosyl-1,8-diamino-3-thio-octane (AdoDATO) and trans-4-methylcyclohexylamine (4MCHA). The results show that binding of dcAdoMet to pfSPDS stabilizes the conformation of the flexible gatekeeper loop of the enzyme and affects the conformation of the active-site amino acid residues, preparing the protein for binding of the second substrate. The complexes of AdoDATO and 4MCHA with pfSPDS reveal the mode of interactions of these compounds with the enzyme. While AdoDATO essentially fills the entire active-site pocket, 4MCHA only occupies part of it, which suggests that simple modifications of this compound may yield more potent inhibitors of pfSPDS.     

外源亚精胺对盐碱胁迫下番茄幼苗根系线粒体功能的影响

为探讨外源亚精胺(Spd)对盐碱胁迫下番茄根系线粒体功能的影响,采用水培法,以耐性不同的两个番茄品种‘金棚朝冠’（耐盐型）和‘中杂9号’（敏感型）为试材,通过模拟盐碱生态条件(NaCl∶Na₂SO₄∶NaHCO₃∶Na₂CO₃=1∶9∶9∶1),结合叶面喷施外源0.25 mmol·L^-1Spd,研究盐碱胁迫8 d后Spd对番茄幼苗根系形态和根系线粒体功能的影响.结果表明： 盐碱胁迫下,两个品种番茄根系线粒体内H₂O₂和丙二醛（MDA）含量增加,线粒体膜通透性明显增大,流动性降低,膜电位、线粒体内细胞色素c/a(Cyt c/a)吸光度比值、膜H⁺-ATPase活性显著下降,使线粒体受到不同程度的损伤,从而抑制根系生长,且‘金棚朝冠’的上述指标变化幅度均小于‘中杂9号’.盐碱胁迫下,喷施外源Spd处理的两个品种根系线粒体H₂O₂和MDA含量显著降低,膜通透性减小、流动性增加,膜电位、线粒体内Cyt c/a吸光度比值、膜H⁺-ATPase活性显著提高,可有效缓解盐碱胁迫对番茄幼苗根系线粒体的伤害作用,且这种缓解作用在‘中杂9号’上的表现效果更佳.     

外源亚精胺对NaCl胁迫下毕氏海蓬子光合参数和叶绿体超微结构的影响

在0、100、300、500和700 mmol·L-1NaCl胁迫条件下比较了喷施0.1mmol·L-1亚精胺(Spd)对毕氏海蓬子(Salicomia bigelovii Torr.)幼苗叶绿素含量、净光合速率、气孔导度、胞间CO2浓度和叶绿体超微结构的影响.结果表明:毕氏海蓬子的叶绿素含量、净光合速率和气孔导度均呈低浓度条件下(0、100和300 mmol·L-1NaCl)升高、高浓度条件下(500和700 mmol·L-1NaCl)降低的趋势,在300 mmol·L-1 NaCl胁迫条件下达到最高值:胞间CO2浓度则呈低浓度NaCl胁迫条件下降低、500 mmol·L-1NaCl条件下升高、700 mmol·L-1NaCl条件下略降低的趋势;在0～500 mmol·L-1NaCl胁迫条件下叶绿素a/b值变化不明显,但在700 mmol·L-1NaCl条件下急剧降低.在低浓度NaCl胁迫条件下,叶绿体整体膨胀,类囊体片层结构松散,但叶绿体和类囊体结构仍保持完整;而经500和700mmol·L-1NaCl处理后,叶绿体超微结构被严重破坏,叶绿体膜结构破裂、类囊体结构松散呈放射状、有些叶绿体完全解体.而在相应的NaCl胁迫条件下喷施0.1 mmol·L-1Spd,毕氏海蓬子的叶绿素含量、净光合速率、气孔导度和胞间CO2浓度虽然也呈现出相同的变化趋势,但其数值均显著高于对照(未喷施Spd);且叶绿体超微结构的损伤程度也轻于对照.研究结果说明:喷施外源Spd能够减缓NaCl胁迫对毕氏海蓬子的伤害作用.     

Effects of Exogenous Spermidine on Antioxidant System Responses of Typha latifolia L. Under Cd2+ Stress

The effects of foliar spraying with spermidine (Spd), ranging in concentration from 0.25 to 0.50mmol/L, on the antioxidant system under Cd2 stress (range 0.1- 0.2 mmol/L Cd2 ) in Typha latifolia L.grown hydroponically were investigated in order to offer a referenced evidence for an understanding of the mechanism by which polyamines (PAs) relieve the damage to plants by heavy metal and improve the phytoremediation efficiency of heavy metal-contaminated water. The results showed that Cd2 stress inhydrogen peroxide (H2O2) and malondialdehyde (MDA) contents in both leaves and caudices. With theexception of superoxide dismutase (SOD) activity in the leaves, an increase in the activities of catalase (CAT), guaiacol peroxidase (GPX), and glutathione reductase (GR) was observed in both leaves and caudices,SOD activity was increased in caudices, and ascorbate peroxidase (APX) activity was increased in leaves following Cd2 treatment. The reduced glutathione (GSH) content in both leaves and caudices and the reductive ascorbate content in leaves was obviously increased, which were prompted by the application of exogenous Spd. Spraying with Spd increased the activity of GR and APX in both leaves and caudices,whereas the activity of SOD, CAT, and GPX was increased only in caudices following spraying with Spd.with Spd. The decrease in MDA was more obvious following the application of 0.25 than 0.50 mmol/L Spd.It is supposed that exogenous Spd elevated the tolerance of T. latifolia under Cd2 stress primarily by increasing GR activity and the GSH level.     

外源多胺对铜胁迫下水鳖叶片多胺代谢、抗氧化系统和矿质营养元素的影响

采用营养液水培的方法,研究了外源亚精胺(Spd)和精胺(Spm)对Cu胁迫下水鳖叶片3种形态多胺(PAs)、抗氧化系统及营养元素的影响。结果表明:(1)Cu胁迫使水鳖叶片腐胺(Put)急剧积累,Spd和Spm明显下降,从而使(Spd+Spm)/Put比值也随之下降。外源Spd和Spm显著或极显著逆转Cu诱导的PAs变化,抑制Put的积累,缓解Spd和Spm的下降,从而提高了(Spd+Spm)/Put比值。(2)外源Spd和Spm抑制了Cu胁迫诱导的多胺氧化酶(PAO)的增加,缓解了二胺氧化酶(DAO)的下降。(3)与单一Cu胁迫相比,Spd和Spm显著或极显著提高了超氧化物歧化酶(SOD)、过氧化物酶(POD)、过氧化氢酶(CAT)、抗坏血酸过氧化物酶(APX)和谷胱甘肽还原酶(GR)活性和抗坏血酸(AsA)、谷胱甘肽(GSH)、游离脯氨酸(Pro)含量,从而降低了超氧阴离子(O2.-)产生速率和过氧化氢(H2O2)含量,极显著降低了丙二醛(MDA)含量,缓解了Cu诱导的氧化胁迫。(4)外源Spd和Spm显著或极显著缓解了Cu胁迫下矿质营养元素吸收平衡的紊乱。以上结果均说明了外施Spd和Spm可增加水鳖对Cu胁迫的耐受性。     

Biosynthesis of spermidine, a direct precursor of pyrrolizidine alkaloids in root cultures of Senecio vulgaris L.

 The polyamine spermidine is an essential biosynthetic precursor of pyrrolizidine alkaloids. It provides its aminobutyl group which is transferred to putrescine yielding homospermidine, the specific building block of the necine base moiety of pyrrolizidine alkaloids. The enzymatic formation of spermidine was studied in relation to the unique role of this polyamine as an alkaloid precursor. S-adenosylmethionine decarboxylase (SAMDC, EC 4.1.1.50) and spermidine synthase (SPDS, EC 2.5.1.16) from root cultures of Senecio vulgaris were partially purified and characterized. The SAMDC-catalyzed reaction showed a pH optimum of 7.5, that of SPDS an optimum of 7.7. The K _m value of SAMDC for its substrate S-adenosylmethionine (SAM) was 15 μM, while the apparent K _m values of SPDS for its substrates decarboxylated SAM (dSAM) and putrescine were 4 μM and 21 μM, respectively. The relative molecular masses of the two enzymes, determined by gel filtration, were 29 000 (SAMDC) and 37 000 (SPDS). Studies with various potential inhibitors revealed, for most inhibitors, profiles that were similar to those established with the respective enzymes from other plant sources. However, putrescine which is not known to be an inhibitor of plant SAMDC, strongly inhibited the enzyme from S. vulgaris roots. Spermidine synthase was sensitive to inhibition by its product spermidine. In the presence of the stationary tissue concentrations of the two polyamines (ca. 0.1 mM each) the activities of SAMDC and SPDS would be inhibited by >80%. The results are discussed in relation to the role of spermidine in primary and secondary metabolism of alkaloid-producing S. vulgaris root cultures. Received: 15 September 1999 / Accepted 10 December 1999     

Spermidine promotes stress resistance in Drosophila melanogaster through autophagy-dependent and -independent pathways

The naturally occurring polyamine spermidine (Spd) has recently been shown to promote longevity across species in an autophagy-dependent manner. Here, we demonstrate that Spd improves both survival and locomotor activity of the fruit fly Drosophila melanogaster upon exposure to the superoxide generator and neurotoxic agent paraquat. Although survival to a high paraquat concentration (20 mM) was specifically increased in female flies only, locomotor activity and survival could be rescued in both male and female animals when exposed to lower paraquat levels (5 mM). These effects are dependent on the autophagic machinery, as Spd failed to confer resistance to paraquat-induced toxicity and locomotor impairment in flies deleted for the essential autophagic regulator ATG7 (autophagy-related gene 7). Spd treatment did also protect against mild doses of another oxidative stressor, hydrogen peroxide, but in this case in an autophagy-independent manner. Altogether, this study establishes that the protective effects of Spd can be exerted through different pathways that depending on the oxidative stress scenario do or do not involve autophagy.