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.     

Metabolic regulation of polyamines and γ‐aminobutyric acid in relation to spermidine‐induced heat tolerance in white clover

• Heat stress decreases crop growth and yield worldwide. Spermidine (Spd) is a small aliphatic amine and acts as a ubiquitous regulator for plant growth, development and stress tolerance.
• Objectives of this study were to determine effects of exogenous Spd on changes in endogenous polyamine (PA) and γ‐aminobutyric acid (GABA) metabolism, oxidative damage, senescence and heat shock protein (HSP) expression in white clover subjected to heat stress. Physiological and molecular methods, including colorimetric assay, high performance liquid chromatography and qRT‐PCR, were applied.
• Results showed that exogenous Spd significantly alleviated heat‐induced stress damage. Application of Spd not only increased endogenous putrescine, Spd, spermine and total PA accumulation, but also accelerated PA oxidation and improved glutamic acid decarboxylase activity, leading to GABA accumulation in leaves under heat stress. The Spd-pretreated white clover maintained a significantly higher chlorophyll (Chl) content than untreated plants under heat stress, which could be related to the roles of Spd in up‐regulating genes encoding Chl synthesis (PBGD and Mg‐CHT) and maintaining reduced Chl degradation (PaO and CHLASE) during heat stress. In addition, Spd up‐regulated HSP70, HSP70B and HSP70‐5 expression, which might function in stabilizing denatured proteins and helping proteins to folding correctly in white clover under high temperature stress.
• In summary, exogenous Spd treatment improves the heat tolerance of white clover by altering endogenous PA and GABA content and metabolism, enhancing the antioxidant system and HSP expression and slowing leaf senescence related to an increase in Chl biosynthesis and a decrease in Chl degradation during heat stress.

     

Comparative proteomic analysis reveals the positive effect of exogenous spermidine on photosynthesis and salinity tolerance in cucumber seedlings

Key message

Our results based on proteomics data and physiological alterations proposed the putative mechanism of exogenous Spd enhanced salinity tolerance in cucumber seedlings.

Abstract

Current studies showed that exogenous spermidine (Spd) could alleviate harmful effects of salinity. It is important to increase our understanding of the beneficial physiological responses of exogenous Spd treatment, and to determine the molecular responses underlying these responses. Here, we combined a physiological analysis with iTRAQ-based comparative proteomics of cucumber (Cucumis sativus L.) leaves, treated with 0.1 mM exogenous Spd, 75 mM NaCl and/or exogenous Spd. A total of 221 differentially expressed proteins were found and involved in 30 metabolic pathways, such as photosynthesis, carbohydrate metabolism, amino acid metabolism, stress response, signal transduction and antioxidant. Based on functional classification of the differentially expressed proteins and the physiological responses, we found cucumber seedlings treated with Spd under salt stress had higher photosynthesis efficiency, upregulated tetrapyrrole synthesis, stronger ROS scavenging ability and more protein biosynthesis activity than NaCl treatment, suggesting that these pathways may promote salt tolerance under high salinity. This study provided insights into how exogenous Spd protects photosynthesis and enhances salt tolerance in cucumber seedlings.

     

Temperature-Stress-Induced Impairment of Chlorophyll Biosynthetic Reactions in Cucumber and Wheat

Chlorophyll (Chl) biosynthesis in chill (7°C)- and heat (42°C)-stressed cucumber (Cucumis sativus L. cv poinsette) seedlings was affected by 90 and 60%, respectively. Inhibition of Chl biosynthesis was partly due to impairment of 5-aminolevulinic acid biosynthesis both in chill- (78%) and heat-stress (70%) conditions. Protochlorophyllide (Pchlide) synthesis in chill- and heat-stressed seedlings was inhibited by 90 and 70%, respectively. Severe inhibition of Pchlide biosynthesis in chill-stressed seedlings was caused by inactivations of all of the enzymes involved in protoporphyrin IX (Proto IX) synthesis, Mg-chelatase, and Mg-protoporphyrin IX monoester cyclase. In heat-stressed seedlings, although 5-aminolevulinic acid dehydratase and porphobilinogen deaminase were partially inhibited, one of the porphyrinogen-oxidizing enzymes, uroporphyrinogen decarboxylase, was stimulated and coproporphyrinogen oxidase and protoporphyrinogen oxidase were not substantially affected, which demonstrated that protoporphyrin IX synthesis was relatively more resistant to heat stress. Pchlide oxidoreductase, which is responsible for phototransformation of Pchlide to chlorophyllide, increased in heat-stress conditions by 46% over that of the control seedlings, whereas it was not affected in chill-stressed seedlings. In wheat (Triticum aestivum L. cv HD2329) seedlings porphobilinogen deaminase, Pchlide synthesis, and Pchlide oxidoreductase were affected in a manner similar to that of cucumber, suggesting that temperature stress has a broadly similar effect on Chl biosynthetic enzymes in both cucumber and wheat.     

Effect of root-applied spermidine on growth and respiratory metabolism in roots of cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis>) seedlings under hypoxia

The effects of exogenous spermidine (Spd) application to hypoxic nutrient solution on the contents of endogenous polyamines (PAs) and respiratory metabolism in the roots of cucumber (Cucumis sativus L.) seedlings were investigated. Cucumber seedlings were grown hydroponically in control and hypoxic nutrient solutions with and without addition of Spd at a concentration of 0.05 mM. The activities of key enzymes involved in the tricarboxylic acid cycle (TCAC), such as succinate dehydrogenase (SDH) and isocitrate dehydrogenase (IDH), were significantly inhibited under root-zone hypoxia with dissolved oxygen (DO) at 1 mg/l. In contrast, the activities of enzymes involved in the process of fermentation, such as pyruvate decarboxylase (PDC), alcohol dehydrogenase (ADH), lactate dehydrogenase (LDH), and alanine aminotransferase (AlaAT), were significantly increased. Thus, aerobic respiration was inhibited and fermentation was enhanced in the roots of cucumber seedlings as a result of decreasing ATP content to inhibit the dry weight of seedlings under hypoxic stress. Moreover, the contents of free, soluble conjugated, and insoluble bound putrescine (Put), Spd, and spermine (Spm) in the roots of cucumber seedlings were significantly increased under hypoxia stress. Interestingly, application of Spd to hypoxic roots markedly suppressed the accumulation of free Put and, in contrast, promoted an increase in free Spd and Spm, as well as soluble conjugated and insoluble bound Put, Spd, and Spm contents. From these data, we deduced that exogenous Spd promotes the conversion of free Put into free Spd and Spm, and soluble conjugated and insoluble bound PAs under hypoxia stress. Furthermore, the activities of LDH, PDC, and ADH were suppressed and, in contrast, the activities of SDH and IDH were enhanced by application of exogenous Spd to hypoxic roots. As a result, aerobic respiration was enhanced but fermentation metabolism was inhibited in the roots of cucumber seedlings, leading to an increase in ATP content to alleviate the inhibited dry weight of seedlings due to hypoxia stress. These results suggest that application of Spd to hypoxic nutrient solution promoted conversion of free Put into free Spd and Spm as well as soluble conjugated and insoluble bound PAs, further enhanced IDH and SDH activities, and inhibited ethanol fermentation and lactate fermentation, resulting in increased ATP content and eventually enhanced tolerance of cucumber plants to root-zone hypoxia.     

外源亚精胺对盐胁迫下黄瓜植株氮化合物含量和硝酸还原酶活性的影响

研究了外源亚精胺(Spd)在营养液栽培中,对盐胁迫下耐盐性不同的两品种黄瓜幼苗体内硝态氮、铵态氮、脯氨酸(Pro)含量和硝酸还原酶(NR)活性的影响。结果表明,外源Spd显著减小了盐胁迫引起的铵态氮、Pro含量的升高幅度和NR活性、硝态氮含量的降低幅度,且对盐敏感型黄瓜品种影响幅度较大。表明Spd可明显减缓盐胁迫对黄瓜幼苗氮素营养代谢的影响。     

Exogenous Spermidine Modulates Osmoregulatory Substances and Leaf Stomata to Alleviate the Damage to Lettuce Seedlings Caused by High Temperature Stress

Lettuce (Lactuca sativa L.) prefers cool environments, and high temperatures affect its yield and quality. Polyamines (PAs) have a mitigating effect on plant abiotic stresses. The effect of exogenous spermidine (Spd) on the osmoregulatory substances and stomata of seedlings of the non-heat-tolerant lettuce variety ‘Bei San 3’ under high temperature stress was investigated at 35 °C/30 °C (day/night) under spray treatment with Spd. The results showed that exogenous Spd increased the total fresh weight, root-to-shoot ratio, leaf length, leaf width, root volume, and root surface area of lettuce under high temperature stress and reduced levels of malondialdehyde. The endogenous polyamine content was changed, and endogenous spermidine (Spd) and putrescine (Put) were increased. The accumulation of six organic osmoregulatory substances was promoted, resulting in enhanced betaine aldehyde dehydrogenase (BADH), choline monooxygenase (CMO), proline catalase pyrroline-5-carboxylate synthase (P5CS), ornithine aminotransferase (OAT), and pyrroline-5-carboxylate reductase (P5CR) activity. The production and activity of the degrading enzymes proline dehydrogenase (PDH) and proline oxidase (POX) were inhibited, and the activity of glutamic acid decarboxylase (GAD), the key enzyme of γ-aminobutyric (GABA), was suppressed. In addition, exogenous Spd increased the contents of Ca, K, Fe, Mn, Zn, and NO₃^? ions in lettuce leaves under high temperature stress, promoted K⁺ efflux and Ca²⁺ influx, and reduced the relative stomatal aperture. In summary, exogenous Spd mitigates lettuce injury caused by high temperature stress by increasing the content of osmoregulatory substances and altering stomatal morphology.

     

Salt‐stress induced modulation of chlorophyll biosynthesis during de‐etiolation of rice seedlings

Chlorophyll biosynthesis in plants is subjected to modulation by various environmental factors. To understand the modulation of the chlorophyll (Chl) biosynthesis during greening process by salt, 100–200 mM NaCl was applied to the roots of etiolated rice seedlings 12 h prior to the transfer to light. Application of 200 mM NaCl to rice seedlings that were grown in light for further 72 h resulted in reduced dry matter production (–58%) and Chl accumulation (–66%). Ionic imbalance due to salinity stress resulted in additional downregulation (41–45%) of seedling dry weight, Chl and carotenoid contents over and above that of similar osmotic stress induced by polyethylene glycol. Downregulation of Chl biosynthesis may be attributed to decreased activities of Chl biosynthetic pathway enzymes, i.e. 5‐aminolevulinic acid (ALA) dehydratase (EC‐2.4.1.24), porphobilinogen deaminase (EC‐4.3.1.8), coproporphyrinogen III oxidase (EC‐1.3.3.3), protoporphyrinogen IX oxidase (EC‐1.3.3.4), Mg‐protoporphyrin IX chelatase (EC‐6.6.1.1) and protochlorophyllide oxidoreductase (EC‐1.3.33.1). Reduced enzymatic activities were due to downregulation of their protein abundance and/or gene expression in salt‐stressed seedlings. The extent of downregulation of ALA biosynthesis nearly matched with that of protochlorophyllide and Chl to prevent the accumulation of highly photosensitive photodynamic tetrapyrroles that generates singlet oxygen under stress conditions. Although, ALA synthesis decreased, the gene/protein expression of glutamyl‐tRNA reductase (EC‐1.2.1.70) increased suggesting it may play a role in acclimation to salt stress. The similar downregulation of both early and late Chl biosynthesis intermediates in salt‐stressed seedlings suggests a regulatory network of genes involved in tetrapyrrole biosynthesis.     

外源褪黑素对高温胁迫条件下黄瓜幼苗氮代谢的影响

以‘津优4号’（热敏感型）和‘美国保尔’（耐热型）黄瓜幼苗为试材,研究了叶面喷施褪黑素对高温胁迫条件下黄瓜幼苗氮代谢的影响。结果显示,高温胁迫下,（1）两种黄瓜幼苗硝态氮含量先升高后降低,‘津优4号’总氮和氨态氮含量先下降后持续升高,而‘美国保尔’总氮和氨态氮含量持续上升;（2）两种黄瓜幼苗硝酸还原酶（NR）活性均先上升后下降,而谷氨酰胺合成酶（Gs）、谷氨酸合成酶（GOGAT）和谷氨酸脱氢酶（GDH）均持续下降,‘美国保尔’的4种酶活性下降幅度显著低于‘津优4号’。研究结果表明,叶面喷施褪黑素可有效缓解高温胁迫对NR、GS、GOGAT和GDH的抑制作用,显著增加硝态氮含量,降低氨态氮含量,减轻氨态氮积累对黄瓜幼苗造成的毒害作用,增强高温胁迫条件下黄瓜幼苗氮素的代谢能力,减轻高温胁迫对黄瓜幼苗造成的伤害,提高黄瓜幼苗抵御高温胁迫的能力。     

外源Spd对高温胁迫下黄瓜幼苗叶片膜脂过氧化及质子泵活性的影响

以较为耐热的黄瓜品种‘津春4号’为试材,在人工气候箱中,采用石英砂培养加营养液浇灌的栽培方式,研究了叶面喷施外源亚精胺(Spd)对高温胁迫下黄瓜幼苗叶片膜脂过氧化程度、质子泵活性及其基因表达的影响.结果表明:高温胁迫下,外源Spd促进黄瓜幼苗株高、茎粗、干、鲜质量和叶面积显著增加,有效抑制叶片相对电导率、丙二醛(MDA)含量和脂氧合酶(LOX)活性的升高,有助于提高叶片细胞质膜和液泡膜H+ -ATPase活性,但在基因表达水平上无显著差异.外源Spd可显著降低黄瓜幼苗叶片膜脂过氧化程度,提高质子泵活性,从而稳定膜的结构和功能,缓解高温胁迫对黄瓜幼苗造成的伤害,提高幼苗对高温胁迫的耐性.     

Spermidine pretreatment enhances heat tolerance in rice seedlings through modulating antioxidative and glyoxalase systems

This study was undertaken to investigate the possible involvement of the antioxidant defense and glyoxalase systems in protecting rice seedlings from heat-induced damage in the presence of spermidine (Spd). Hydroponically grown 14-day-old seedlings were subjected to foliar spray with Spd (1 mM, 24 h) prior to heat stress (42 °C, 48 h) followed by subsequent recovery (27 °C, 48 h). Lipoxygenase activity, malondialdehyde (MDA), hydrogen peroxide (H₂O₂) and proline (Pro) content increased significantly whereas fresh weight (FW) and chlorophyll (Chl) content decreased during heat stress and after recovery, indicating unrecoverable damage to rice seedlings. Heat-induced damage was also evident in decreased levels of ascorbate (AsA), glutathione (GSH), and AsA and GSH redox ratios. Superoxide dismutase (SOD) and catalase (CAT) activities increased during heat stress but declined after recovery. Activities of glutathione peroxidase (GPX), ascorbate peroxidase (APX), monodehydroascorbate reductase, dehydroascorbate reductase (DHAR) and glutathione reductase (GR) decreased during heat stress but an opposite trend for most of these enzymes was observed after recovery. Heat stress also resulted in significant increases in the activities of glyoxalase enzymes (Gly I and Gly II). In contrast, exogenous Spd protected rice seedlings from heat-induced damage as marked by lower levels of MDA, H₂O₂, and Pro content coupled with increased levels of AsA, GSH, FW, Chl, and AsA and GSH redox status. After recovery, Spd-pretreated heat-exposed seedlings displayed higher activities of SOD, CAT, GPX, GST APX, DHAR and GR as well as of Gly I and Gly II. In addition, polyamine analysis revealed that exogenously applied Spd significantly elevated the levels of free and soluble conjugated Spd. Therefore, we conclude from our results that heat exposure provoked an oxidative burden while enhancement of the antioxidative and glyoxalase systems by Spd rendered rice seedlings more tolerant to heat stress. Further, co-induction of the antioxidative and glyoxalase systems was closely associated with Spd mediated enhanced level of GSH.     

Effect of low dose of spermidine on physiological changes in salt-stressed cucumber plants

The present study was aimed to assess the ameliorative potentiality of exogenously applied low dose of spermidine (Spd) (4.0 mL of 0.1 mM) against salt stress in cucumber (Cucumis sativus L.) plants. Salt stress inhibited plant growth, while Spd increased the shoot length and dry weight of leaves in salt-stressed plants. Chlorophyll, carotenoids, and sucrose contents were lower, and the accumulation of superoxide radical was higher in salt-affected plants than in controls, and these detrimental effects were mitigated by Spd treatment. Moreover, salinity diminished the reduced glutathione and total polyphenols and inhibited the activities of catalase, peroxidase, and polyphenol oxidase as compared with controls, and Spd treatment increased all antioxidant activities in salt-injured plants. NaCl-induced oxidative stress caused a significant decrease in GA₄ and GA₅ contents. Spd treatment ameliorated these salt stress effects by increasing the quantities of GA₄. In addition, sodium content was higher and calcium content was lower in salt-treated plants, while Spd treatment reduced the sodium accumulation and increased the calcium level in plants exposed to NaCl. The results suggest that exogenous application of low Spd dose can ameliorate the salt stress effects on cucumber by modulating the components of photosynthetic pigments, antioxidants, gibberellins, and minerals.     

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。     

外源脯氨酸对高温胁迫下黄瓜幼苗叶片AsA-GSH循环和光合荧光特性的影响

以抗热性较弱的黄瓜品种‘新泰密刺'为试材,在人工气候箱内采用营养液栽培法,研究了外源脯氨酸(Pro)预处理对高温胁迫下黄瓜幼苗叶片抗坏血酸-谷胱甘肽循环和光合荧光特性的影响.结果显示:(1)与清水处理相比,高温胁迫4 h和8 h时,Pro预处理黄瓜幼苗叶片单脱氢抗坏血酸还原酶(MDAR)活性、GSH(还原型谷胱甘肽)/GSSG(氧化型谷胱甘肽)比值及GSH含量显著升高;(2)在高温胁迫8 h时,Pro预处理幼苗的净光合速率(P_n)、气孔导度(G_s)及PSⅡ的最大光化学效率(F_v/F_m)、光化学淬灭系数(q_P)均显著升高,而蒸腾速率(T_r)和非光化学淬灭系数(NPQ)降低.研究表明,外源Pro预处理可显著提高高温胁迫下黄瓜幼苗叶片抗坏血酸-谷胱甘肽循环清除H_2O_2能力和叶片光合能力,有效缓解高温胁迫对黄瓜叶片抗氧化系统和光合系统的伤害,从而增强植株的耐热性.