Exogenous spermidine-induced changes at physiological and biochemical parameters levels in tomato seedling grown in saline-alkaline condition
© Zhang et al.; licensee Springer 2014
Received: 25 May 2014
Accepted: 21 July 2014
Published: 1 August 2014
Tomato is one of the most popular vegetables, and middle tolerance for salt stress. Spermidine (Spd) has an important role in plant defense mechanisms against abiotic stress; however, relatively few data are available regarding Spd in responses of tomato to saline-alkaline stress. The effect of 0.25 mmol/L Spd on some physiological parameters of two tomato cultivars grown in 75 mmol/L saline-alkaline solutions were studied. Two cultivars are cv. Jinpeng chaoguan which is a highly salt-tolerant ecotype and cv. Zhongza No. 9 which is more salt-sensitive ecotype.
Saline-alkaline stress upset nitrogen metabolism, induced the antioxidant enzyme activities, and accumulated much more reactive oxygen species (ROS) and osmoregulation substances in two tomato cultivars leaves. Under saline-alkaline stress condition, Spd-treated seedlings accumulated more osmoregulation substances and had greater activities of antioxidative enzymes. Exogenous Spd counteracted the stress-induced increase of contents of malondialdehyde and ammonium, glutamate dehydrogenase activity, and decreased in nitrate, nitrate reductase, nitrite reductase, glutamine synthetase, glutamate synthase, glutamate oxaloacetate transaminase, and glutamate pyruvate transaminase activities. Additionally, the effect of Spd was more significantly in salt-sensitive cultivar ‘Zhongza No. 9’.
Overall, exogenous spermidine can attenuate negative effects of saline-alkaline stress on tomato seedlings which effects may depend on the plant species, and even cultivars.
KeywordsTomato Saline-alkaline stress Spermidine Nitrogen metabolism Antioxidant enzyme Osmoregulation substance
Saline-alkaline condition imposes a major abiotic stress on crops and represents an important limiting factor of productivity. It has been estimated that one-third of the world’s irrigated land is unsuitable for crops due to its saline condition (Frommer et al. ; Wasti et al. ). Plant responses to mixed salt and alkali stress are more complex than their responses to either simple salt or alkali stress (Shi and Sheng ; Yang et al. ) and there have been few studies of complex neutral and alkaline salt stress. In general, plant metabolism is altered and a range of defense mechanisms are activated in response to abiotic stress, presumably to compensate for the changed environmental conditions (Wasti et al. ). Stress could induce excessive generation of reactive oxygen species (ROS) including superoxide anion (O2−), hydrogen peroxide (H2O2) and hydroxyl radical (HO-), which could cause deterioration of membrane lipids, proteins and nucleic acids, leading to increased membrane leakage of solutes (Shehab et al. ; Huang et al. ). One stress-defense mechanism in plants is the accumulation of compatible osmolytes (Shahba et al. ), which can also be induced or enhanced by the application of chemicals to the plant (Rhodes et al. ).
Polyamines are low-molecular-weight aliphatic amines with important functions in growth, cell division, DNA replication, and protein synthesis (Roychoudhury et al. ). Spermidine (Spd), spermine, and putrescine are major polyamines in plants that act as second messengers, mediating responses to various environmental stressors. These stressors include osmotic stress, changes in salinity, drought conditions, and exposure to ozone, heavy metals, and ultraviolet light (Groppa and Benavides ). Compared with other type of polyamine, Spd could more effectively alleviate the adverse impacts of salinity-alkalinity (Hu et al., ).
Despite research efforts, little is known about the physiological functions of exogenous Spd in response to salt-alkali mixed stress. The objective of this work was to determine effects of Spd under saline-alkaline conditions in two tomato cultivars with different salinity tolerance. The response in terms of nitrogen metabolism, antioxidant enzyme activities, contents of ROS and osmoregulation substances were evaluated in order to evaluate the role of Spd in promoting tomato plants tolerance to saline-alkaline stress.
Plant materials and treatments
Two tomato (Solanum lycopersicum) cultivars were used in the study. cv. Jinpeng chaoguan is a highly salt-tolerant ecotype, while cv. Zhongza No. 9 is more salt-sensitive ecotype (Hu et al. ). Tomato seeds were surface-sterilized with 4% (v/v) sodium hypochlorite, rinsed with distilled water, soaked in distilled water for 6 h at 26°C, and transferred to sterile moist Whatman No. 1 filter paper which moistened with distilled water in Petri plates. The plates were maintained in the dark at 26°C for germination. Uniformly germinated seeds were selected and cultivated in polystyrene trays filled with complex organic substrates and placed in a greenhouse with an average temperature of 26-30°C during the day and 16-18°C at night, a 16-h light (600-800 μmol · photons/m2 · s) followed by an 8-h dark photoperiod, and a 50-90% relative humidity. When the third leaves were fully expanded, we transplanted all of the seedlings into rectangular hydroponic containers containing continuously aerated half-strength Hoagland’s nutrient solution. When tomato seedlings were at the sixth true leaves stage, the seedlings were treated with the following treatments: (1) control (CK), half-strength Hoagland’s nutrient solution cultivation, (b) saline-alkaline treatment, tomato seedlings were exposed to half-strength Hoagland’s nutrient solution cultivation contain 75 mmol/L saline-alkaline solution (NaCl:Na2SO4:NaHCO3:Na2CO3 = 1:9:9:1), (c) saline-alkaline plus Spd treatment, tomato seedlings were exposed to half-strength Hoagland’s nutrient solution cultivation contain 75 mmol/L saline-alkaline solution and sprayed with 0.25 mmol/L Spd (Sigma-Aldrich, St. Louis, MO, USA). The experiment took place in a greenhouse covered totally with polycarbonate sheets and located at the horticultural experimental station of Northwest Agriculture & Forestry University, China. The experiment design included 3 treatments of each cultivar, totally being 6 treatments with 3 times under the same conditions. Each treatment has 3 containers, and each container includes 12 plants every time. The sixth leaves were harvested 4 days after the treatment for analysis different indexes.
Analyses of NO3−-N and NH4+-N levels
Leaf samples were dried at 75°C until constant weight was obtained. The dried material (200 mg) was ground to a powder and extracted in 10 mL of distilled water for 2.5 h. Contents of NO3−-N and NH4+-N were determined according to the method of Cataldo et al. () and Krom (), respectively.
Assays of nitrogen metabolism enzymes
Nitrate reductase (NR), nitrite reductase (NiR), Glutamate dehydrogenase (GDH) and Glutamate synthetase (GOGAT) activities were estimated according to the methods of Gangwar and Singh (). Glutamine synthetase (GS) activity was measured using an adaptation of Lillo’s method (Lillo ). The activities of glutamate oxaloacetate transaminase (GOT) and glutamate pyruvate transaminase (GPT) were measured with the method described by Liang et al. ().
Antioxidant enzyme activity, H2O2,O2− and malondialdehyde assay
Superoxide dismutase (SOD) activity was assayed as previously described by Dhindsa et al. (). One unit of activity was defined as the amount of enzyme required to inhibit the reduction of nitro blue tetrazolium chloride by 50% at 560 nm. Peroxidase (POD) activity was assayed as previously described (Kochba et al. ). One unit of activity was defined as the amount of enzyme required to increase absorbance by 0.1 absorbance units at an optical density of 470 nm per min. Catalase (CAT) activity was assayed as described by Dhindsa et al. (). One unit of activity was defined as the amount of enzyme required to decrease 0.1 absorbance units at an optical density of 240 nm per min. Contents of H2O2 and O2− were detected in leaves as described by Orozco-Cardenas et al. (). The content of malondialdehyde (MDA) was measured according to the method of Xu et al. ().
Measurements of proline, soluble sugar, and soluble protein
The proline content was estimated following the method of Bates et al. (). Soluble sugars were estimated by the anthrone reagent method using glucose as the standard (Yemm and Willis ). Protein was determined according to the method of Bradford () using bovine serum albumin as a standard.
All data presented are the mean values. All experiments were conducted using three replicates at least. All data were statistically analyzed by the analysis of variance (ANOVA) with SAS software (Version 8.1; SAS Institute, Cary, NC, USA)) using Duncan’s multiple range test at the 0.05 level of significance.
Results and discussions
The effect of Spd on NO3−-N and NH4+-N content
The effect of Spd on activities of nitrogen metabolism enzymes
Compared with the only salinity-alkalinity stress, application of Spd significantly promoted NR, GS, GOT, GPT activities in two tomato cultivars, and inhibited GDH activity and had relatively little effect on leaf GOGAT activities in two tomato cultivars (Figure 2). Spd had more effects on nitrogen metabolism enzymes activities in ‘Zhongza No. 9’, except for GOT activity (Figure 2). Changes in forms of nitrogen and the particular complement of metabolic enzymes present can reflect a plant’s adaptation of nitrogen metabolism to stress (Gangwar and Singh, ). The experiment indicates that exogenous Spd partly counteracted stress-induced increases in NH4+-N levels and GDH activity, as well as increased in NO3−-N content and the activities of NR, NiR, GS, GOGAT, and GOT in tomato seedling leaves under saline-alkaline stress. Spd may act as a kind of multi-functional signaling molecule that can activate a variety of defense reactions, resulting in maintenance of normal metabolism and enhanced resistance (Groppa and Benavides ). The positive effects of exogenous Spd on plant nitrogen metabolism may also include further alleviation of saline-alkaline-resulted injuries. The Spd-induced resistance to saline-alkaline stress may be associated with the conversion of Spd to putrescine or spermine. Our previous results showed that exogenous Spd promoted the conversion of free putrescine to free Spd and spermine under salinity-alkalinity stress (Hu et al., ), which suggested that exogenous Spd treatment can regulate the metabolic status of polyamines caused by salinity-alkalinity stress, and eventually enhance tolerance of tomato plants to salinity-alkalinity stress. Besides, some differences in the functions of Spd among cultivars of a given species exist. However, based on our observations, exogenous Spd improves stressed plants more than a control, especially in ‘Zhongza No. 9’ which is a sensitivity cultivar. This result suggested that under saline-alkaline stress, the effects of exogenous Spd on nitrogen metabolism may depend on the plant species, and even cultivars.
Changes in antioxidant enzymes, H2O2 , O2−, and MDA contents in leaves
The effect of Spd on osmoregulation substance
In conclusion, the present results suggested that Spd positively enhanced salinity-alkalinity tolerance in tomato. Its action was associated with nitrate metabolism, antioxidant enzymes and osmoregulation. The accumulation of compatible osmolytes may compensate for the decreased water potential during salinity-alkalinity stress. Additionally, the effect of Spd was more significantly in salt-sensitive cultivar ‘Zhongza No. 9’. Overall, exogenous Spd attenuated negative effects of saline-alkaline stress on plants which effects have varying effects on different tolerant tomato cultivars.
Glutamine 2-oxoglutarate aminotransferase
Glutamate oxaloacetate transaminase
Glutamate pyruvate transaminase
Reactive oxygen species
This work was supported by grants from the National Natural Science Foundation of China (No. 31101581), National Key Technology R&D Program in the 12th Five-year Plan of China (No. 2011BAD29B01), Scientific Research Special Fund of Northwest Agriculture & Forestry University (QN2013018), and China Agriculture Research System (No. CARS-25-D-02).
- Abdel-Latef AA: Salt tolerance of some wheat cultivars. South Valley Univ in Qena, Egypt; 2005.Google Scholar
- Alcázar R, Altabella T, Marco F, Bortolotti C, Reymond M, Koncz C, Carrasco P, Tiburcio AF: Polyamines: molecules with regulatory functions in plant abiotic stress tolerance. Planta 2010, 231: 1237–1249. doi:10.1007/s00425.010.1130.0 doi:10.1007/s00425.010.1130.0 10.1007/s00425-010-1130-0View ArticlePubMedGoogle Scholar
- Bates LS, Wadern RP, Teare ID: Rapid estimation of free proline for water stress determination. Plant Soil 1973, 39: 205–207. doi:10.1007/BF00018060 doi:10.1007/BF00018060 10.1007/BF00018060View ArticleGoogle Scholar
- Bradford MM: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976, 72: 248–254. doi:10.1016/0003–2679(76) 90527–3 doi:10.1016/0003-2679(76) 90527–3 10.1016/0003-2697(76)90527-3View ArticlePubMedGoogle Scholar
- Cataldo DA, Haroon M, Schrader LE, Youngs VL: Rapid colorimetric determination of nitrate in plant tissue by nitration of salicylic acid. Commun Soil Sci Plant 1975, 6: 71–80. doi:10.1080/00103627509366547 doi:10.1080/ 00103627509366547View ArticleGoogle Scholar
- Dev TB, Herbert JK: NH 4 + toxicity in higher plants: a critical review. J Plant Physiol 2002, 159: 567–584. doi:10.1078/0176–1617–0774 doi:10.1078/0176-1617-0774 10.1078/0176-1617-0774View ArticleGoogle Scholar
- Dhindsa RS, Plumb-dhindsa P, Thorpe TA: Leaf senescence: correlated with increased levels of membrane permeability and lipid peroxidation, and decreased levels of superoxide dismutase and catalase. J Exp Bot 1981, 32: 93–101. doi:10.1093/jxb/32.1.93 doi:10.1093/jxb/32.1.93 10.1093/jxb/32.1.93View ArticleGoogle Scholar
- Frommer WB, Ludewig U, Rentsch D: Taking transgenic plants with a pinch of salt. Science 1999, 285: 1222–1223. doi:10.1126/science.285.5431.1222 doi:10.1126/science.285.5431.1222 10.1126/science.285.5431.1222View ArticlePubMedGoogle Scholar
- Gajewska E, Sklodowska M: Nickel-induced changes in nitrogen metabolism in wheat shoots. J. Plant Physiol 2009, 166: 1034–1044. doi:10.1016/j.jplph.2008.12.004 doi:10.1016/j.jplph.2008.12.004View ArticleGoogle Scholar
- Gangwar S, Singh VP (2011) Indole acetic acid differently changes growth and nitrogen metabolism in Pisum sativum L. seedlings under chromium (VI) phytotoxicity: Implication of oxidative stress. Sci Hort 129:321–328. doi:10.1016/j.scienta.2011.03.026 Gangwar S, Singh VP (2011) Indole acetic acid differently changes growth and nitrogen metabolism in Pisum sativum L. seedlings under chromium (VI) phytotoxicity: Implication of oxidative stress. Sci Hort 129:321–328. doi:10.1016/j.scienta.2011.03.026
- Groppa MD, Benavides MP: Polyamines and abiotic stress: recent advances. Amino Acids 2008, 34: 35–45. doi:10.1007/s00726–007–0501–8 doi:10.1007/s00726-007-0501-8 10.1007/s00726-007-0501-8View ArticlePubMedGoogle Scholar
- Hu XH, Zhang Y, Shi Y, Zhang Z, Zou ZR, Zhang H, Zhao JZ: Effect of exogenous spermidine on polyamine content and metabolism in tomato exposed to salinity-alkalinity mixed stress. Plant Physiol Biochem 2012, 57: 200–209. doi:10.1016/j.plaphy.2012.05.015 doi:10.1016/j.plaphy.2012.05.015View ArticlePubMedGoogle Scholar
- Huang CJ, Zhao SY, Wang LC, Anjum SA, Chen M, Zhou HF, Zou CM (2013) Alteration in chlorophyll fluorescence, lipid peroxidation and antioxidant enzymes activities in hybrid ramie (Boehmeria nivea L.) under drought stress. Aust J Crop Sci 7:594–599Google Scholar
- Kochba J, Lavee S, Spiegel-Roy P: Differences in peroxidase activity and isoenzymes in embryogenic and non-embryogenic ‘Shamouti’ orange ovular callus lines. Plant Cell Physiol 1977, 18: 463–467.Google Scholar
- Kong-Ngern K, Daduang S, Wongkham CH, Bunnag S, Kosittrakuna M, Theerakulpisuta P: Protein profiles in response to salt stress in leaf sheaths of rice seedlings. Sci Asia 2005, 31: 403–408. doi:10.2306/scienceasia1513–1874.2005.31.403 doi:10.2306/ scienceasia1513-1874.2005.31.403 10.2306/scienceasia1513-1874.2005.31.403View ArticleGoogle Scholar
- Krom MD: Spectrophotometric determination of ammonia: a study of a modified Berthelot reaction using salicylate and dichloroisocyanurate. Analyst 1980, 105: 305–316. doi:10.1039/AN9800500305 doi:10.1039/AN9800500305 10.1039/an9800500305View ArticleGoogle Scholar
- Liang CG, Chen LP, Wang Y, Liu J, Xu GL, Li T: High temperature at grain-filling stage affects nitrogen metabolism enzyme activities in grains and grain nutritional quality in rice. Rice Sci 2011, 18: 210–216. doi:10.1016/S1672–6308(11)60029 doi:10.1016/S1672-6308(11)60029 10.1016/S1672-6308(11)60029-2View ArticleGoogle Scholar
- Lillo C: Diurnal variations of nitrite reductase, glutamine synthetase, glutamate synthase, alanine aminotransferase and aspartate aminotransferase in barley leaves. Physiol Plant 1984, 61: 214–218. doi:10.1111/j.1399–3054.1984.tb05899.x doi:10.1111/j.1399-3054.1984.tb05899.x 10.1111/j.1399-3054.1984.tb05899.xView ArticleGoogle Scholar
- Masclaux-Daubresse C, Reisdorf-Cren M, Pageau K, Lelandias M, Grandjean J, Valadier MH, Feraud M, Jouglet T, Suzuki A: Glutamine synthetase-glutamate synthase pathway and glutamate dehydrogenase play distinct roles in the sink source nitrogen cycle in tobacco. Plant Physiol 2006, 140: 444–456. doi:10.1104/pp.105.071910 doi:10.1104/pp.105.071910 10.1104/pp.105.071910View ArticlePubMedPubMed CentralGoogle Scholar
- Orozco-Cardenas ML, Narvaez-Vasquez J, Ryan CA: Hydrogen peroxide acts as a second messenger for induction of defense genes in tomato plants in response to wounding, systemin, and methyl jasmonate. Plant Cell 2001, 13: 179–191. doi:10.1105/tpc.13.1.179 doi:10.1105/tpc.13.1.179 10.1105/tpc.13.1.179View ArticlePubMedPubMed CentralGoogle Scholar
- Ramanjulu S, Veeranjaneyulu K, Sudhakar C (1994) Short-term shifts in nitrogen metabolism in mulberry (Morus alba L.) under salt shock. Phytochem 37:991–995. doi:10.1016/S0031–9422(00)89515–1 Ramanjulu S, Veeranjaneyulu K, Sudhakar C (1994) Short-term shifts in nitrogen metabolism in mulberry (Morus alba L.) under salt shock. Phytochem 37:991–995. doi:10.1016/S0031-9422(00)89515-1
- Rhodes D, Verslues PE, Sharp RE: Role of amino acids in abiotic stress resistance. In Plant Amino Acids: Biochemistry and Biotechnology. Edited by: Singh BK. Dekker, New York; 1999:319–356.Google Scholar
- Roychoudhury A, Basu S, Sengupta DN: Amelioration of salinity stress by exogenously applied spermidine or spermine in three varieties of indica rice differing in their level of salt tolerance. J Plant Physiol 2011, 168: 317–328. doi:10.1016/j.jplph.2010.07.009 doi:10.1016/j.jplph.2010.07.009 10.1016/j.jplph.2010.07.009View ArticlePubMedGoogle Scholar
- Shahba Z, Baghizadeh A, Vakili SMA, Yazdanpanah A, Yosefi M (2010) The salicylic acid effect on the tomato (Lycopersicum esculentum Mill.) sugar, protein, and proline contents under salinity stress (NaCl). J Biophys Struct Biol 2:35–41 Shahba Z, Baghizadeh A, Vakili SMA, Yazdanpanah A, Yosefi M (2010) The salicylic acid effect on the tomato (Lycopersicum esculentum Mill.) sugar, protein, and proline contents under salinity stress (NaCl). J Biophys Struct Biol 2:35–41
- Shehab GG, Ahmed OK, El-beltagi HS (2010) Effects of various chemical agents for alleviation of drought stress in rice plants (Oryza sativa L.). Not Bot Hort Agrobot Cluj 38:139–148 Shehab GG, Ahmed OK, El-beltagi HS (2010) Effects of various chemical agents for alleviation of drought stress in rice plants (Oryza sativa L.). Not Bot Hort Agrobot Cluj 38:139–148
- Shi DC, Sheng YM: Effect of various salt–alkaline mixed stresses conditions on sunflower seedlings and analysis of their stress factors. Environ Exp Bot 2005, 54: 8–21. doi:10.1016/j.envexpbot.2004.05.003 doi:10.1016/j.envexpbot.2004.05.003 10.1016/j.envexpbot.2004.05.003View ArticleGoogle Scholar
- Skopelitis DS, Paranychianakis NV, Paschalidis KA: Abiotic stress generates ROS that signal expression of anionic glutamate dehydrogenases to form glutamate for proline synthesis in tobacco and grapevine. Plant Cell 2006, 18: 2767–2781. doi:10.1105/tpc.105.038323 doi:10.1105/tpc.105.038323 10.1105/tpc.105.038323View ArticlePubMedPubMed CentralGoogle Scholar
- Undovenko GV: Effect of salinity of substrateon nitrogen metabolism of plants with different salt tolerance. Agrokhimiya 1971, 3: 23–31.Google Scholar
- Wang ZQ, Yuan YZ, Ou JQ, Lin QH, Zhang CF (2007) Glutamine synthetase and glutamate dehydrogenase contribute differentially to proline accumulation in leaves of wheat (Triticum aestivum) seedlings exposed to different salinity. J Plant Physiol 164:695–701. doi:10.1016/j.jplph.2006.05.001 Wang ZQ, Yuan YZ, Ou JQ, Lin QH, Zhang CF (2007) Glutamine synthetase and glutamate dehydrogenase contribute differentially to proline accumulation in leaves of wheat (Triticum aestivum) seedlings exposed to different salinity. J Plant Physiol 164:695–701. doi:10.1016/j.jplph.2006.05.001
- Wasti S, Mimouni H, Smiti S, Zid E, Ahmed HB: Enhanced salt tolerance of tomatoes by exogenous salicylic acid applied through rooting medium. J Integrative Bio 2012, 16: 200–207. doi:10.1089/omi.2011.0071 doi:10.1089/omi.2011.0071Google Scholar
- Xu PL, Guo YK, Bai JG, Shang L, Wang XJ: Effects of long-term chilling on ultrastructure and antioxidant activity in leaves of two cucumber cultivars under low light. Physiol Plant 2008, 132: 467–478. doi:10.1111/j.1399–3054.2007.01036.x doi:10.1111/j.1399-3054.2007.01036.x 10.1111/j.1399-3054.2007.01036.xView ArticlePubMedGoogle Scholar
- Yang CW, Chong JN, Kim CM, Li CY, Shi DC, Wang DL (2007) Osmotic adjustment and ion balance traits of an alkali resistant halophyte Kochia sieversiana during adaptation to salt and alkali conditions. Plant Soil 294:263–276. doi:10.1007/s11104–007–9251–3 Yang CW, Chong JN, Kim CM, Li CY, Shi DC, Wang DL (2007) Osmotic adjustment and ion balance traits of an alkali resistant halophyte Kochia sieversiana during adaptation to salt and alkali conditions. Plant Soil 294:263–276. doi:10.1007/s11104-007-9251-3
- Yemm EW, Willis AJ: The estimation of carbohydrates in plant extracts by anthrone. Biochem J 1954, 57: 508–514.View ArticlePubMedPubMed CentralGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.