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NO is involved in spermidine-induced drought tolerance in white clover via activation of antioxidant enzymes and genes

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Abstract

Nitric oxide (NO), a key signaling molecule, can be induced by polyamines (PAs), which play an important role in improving drought tolerance in plants. This study was to further investigate the role of NO in spermidine (Spd)-induced drought tolerance associated with antioxidant defense in leaves of white clover (Trifolium repens) under drought stress induced by −0.3 MPa polyethylene glycol (PEG-6000) solution. A hydroponic growth method was used for cultivating plants in a controlled growth chamber for 30–33 days until the second leaves were fully expanded. Two relative independent experiments were carried out in our study. One is that exogenous application of Spd or an NO donor (sodium nitroprusside (SNP)) significantly improved drought tolerance in whole plants, as demonstrated by better phenotypic appearance, increased relative water content (RWC), and decreased electrolyte leakage (EL) and malondialdehyde (MDA) content in leaves as compared to untreated plants. For another detached leaf experiment, PEG induced an increase in the generation of NO in cells and significantly improved activities of nitrate reductase (NR) and nitric oxide synthase (NOS). These responses could be blocked by pre-treatment with a Spd biosynthetic inhibitor, dicyclohexyl amine (DCHA), and then reversed by application of exogenous Spd. Meanwhile, PEG induced up-regulation of activities and gene transcript levels of corresponding antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), and ascorbate peroxidase (APX) to varying degrees, while these effects were partially blocked by pre-treatment with DCHA, the scavenger of NO, the inhibitors of NR or NOS. In addition, Spd-induced antioxidant enzyme activities and gene expression also could be effectively inhibited by an NO scavenger as well as inhibitors of NR and NOS. These findings suggest that both Spd and NO can enhance drought tolerance. Spd was involved in drought stress-activated NR and NOS pathways associated with NO release, which mediated antioxidant defense and thus contributed to drought tolerance in white clover.

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References

  • Amri E, Mohammadi MJ (2012) Effects of timing of drought stress on pomegranate seedlings (Punica granatum L. cv ‘Atabaki’) to exogenous spermidine and putrescine polyamines. Afr J Microbiol Res 6:5294–5300

    CAS  Google Scholar 

  • Arasimowicz-Jelonek M, Floryszak-Wieczorek J, Kubiś J (2009a) Involvement of nitric oxide in water stress-induced responses of cucumber roots. Plant Sci 177:682–690

    Article  CAS  Google Scholar 

  • Arasimowicz-Jelonek M, Floryszak-Wieczorek J, Kubiś J (2009b) Interaction between polyamine and nitric oxide signaling in adaptive responses to drought in cucumber. J Plant Growth Regul 28:177–186

    Article  CAS  Google Scholar 

  • Bais HP, Ravishankar GA (2002) Role of polyamines in the ontogeny of plants and their biotechnological applications. Plant Cell Tissue Organ Cult 69:1–34

    Article  CAS  Google Scholar 

  • Barrs HD, Weatherley PE (1962) A re-examination of the relative turgidity technique for estimating water deficits in leaves. Aust J Biol Sci 15:413–428

    Article  Google Scholar 

  • Blum A, Ebercon A (1981) Cell membrane stability as a measure of drought and heat tolerance in wheat. Crop Sci 21:43–47

    Article  Google Scholar 

  • Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Article  CAS  PubMed  Google Scholar 

  • Chance B, Maehly AC (1955) Assay of catalase and peroxidase. Methods Enzymol 2:764–775

    Article  Google Scholar 

  • Corpas FJ, Barroso JB, Carreras A, Valderrama R, Palma JM, León AM, del Río LA (2006) Constitutive arginine-dependent nitric oxide synthase activity in different organs of pea seedlings during plant development. Planta 224:246–254

    Article  CAS  PubMed  Google Scholar 

  • Corpas FJ, Leterrier M, Valderrama R, Airaki M, Chaki M, Palma JM, Barroso JB (2011) Nitric oxide imbalance provokes a nitrosative response in plants under abiotic stress. Plant Sci 181:604–611

    Article  CAS  PubMed  Google Scholar 

  • Dhindsa RS, Dhindsa PP, Thorpe TA (1981) Leaf senescence: correlated with increased leaves of membrane permeability and lipid peroxidation, and decreased levels of superoxide dismutase and catalase. J Exp Bot 32:93–101

    Article  CAS  Google Scholar 

  • Fan QJ, Liu JH (2012) Nitric oxide is involved in dehydration/drought tolerance in Poncirus trifoliata seedlings through regulation of antioxidant systems and stomatal response. Plant Cell Rep 31:145–154

    Article  CAS  PubMed  Google Scholar 

  • Farooq M, Wahid A, Lee DJ (2009) Exogenously applied polyamines increase drought tolerance of rice by improving leaf water status, photosynthesis and membrane properties. Acta Physiol Plant 31:937–945

    Article  CAS  Google Scholar 

  • Garcia-Mata C, Lamattina L (2001) Nitric oxide induces stomatal closure and enhances the adaptive plant responses against drought stress. Plant Physiol 126:1196–1204

    Article  CAS  PubMed  Google Scholar 

  • Giannopolitis CN, Ries SK (1977) Superoxide dismutase I. Occurrence in higher plants. Plant Physiol 59:309–314

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Hageman RH, Reed AJ (1980) Nitrate reductase from higher plants. Methods Enzymol 23:491–503

    Article  Google Scholar 

  • Hao GP, Xing Y, Zhang JH (2008) Role of nitric oxide dependence on nitric oxide synthase-like activity in the water stress signaling of maize seedling. J Integr Plant Biol 50:435–442

    Article  CAS  PubMed  Google Scholar 

  • He L, Ban Y, Inoue H, Matsuda N, Liu J, Moriguchi T (2008) Enhancement of spermidine content and antioxidant capacity in transgenic pear shoots overexpressing apple spermidine synthase in response to salinity and hyperosmosis. Phytochemistry 69:2133–2141

    Article  CAS  PubMed  Google Scholar 

  • Hevel JM, Marletta MA (1994) Nitric-oxide synthase assays. Methods Enzymol 233:250–258

    Article  CAS  PubMed  Google Scholar 

  • Hoagland DR, Arnon DI (1950) The water culture method for growing plants without soil. Circular. Calif Agric Exp Station 347:1–32

    Google Scholar 

  • Hossain KK, Itoh RD, Yoshimura G, Oku H, Cohen MF, Yamasaki H (2010) Effects of nitric oxide scavengers on thermoinhibition of seed germination in Arabidopsis thaliana. Russ J Plant Physiol Engltr 57:222–232

    Article  CAS  Google Scholar 

  • Hussain SS, Ali M, Ahmad M, Siddique KH (2011) Polyamines: natural and engineered abiotic and biotic stress tolerance in plants. Biotechnol Adv 29:300–311

    Article  CAS  PubMed  Google Scholar 

  • Kolbert Z, Bartha B, Erdei L (2005) Generation of nitric oxide in roots of Pisum sativum, Triticum aestivum and Petroselinum crispum plants under osmotic and drought stress. Acta Biol Szegediensis 49:13–16

    Google Scholar 

  • Kubiś J (2008) Exogenous spermidine differentially alters activities of some scavenging system enzymes, H2O2 and superoxide radical levels in water-stressed cucumber leaves. J Plant Physiol 165:397–406

    Article  PubMed  Google Scholar 

  • Kusano T, Yamaguchi K, Berberich T, Takahashi Y (2007) Advances in polyamine research in 2007. J Plant Res 120:345–350

    Article  CAS  PubMed  Google Scholar 

  • Li Z, Shi P, Peng Y (2013) Improved drought tolerance through drought preconditioning associated with changes in antioxidant enzyme activities, gene expression and osmoregulatory solutes accumulation in white clover (Trifolium repens L.). Plant Omics 6:481–489

    CAS  Google Scholar 

  • Li Z, Peng Y, Zhang XQ, Ma X, Huang LK, Yan YH (2014a) Exogenous spermidine improves seed germination of white clover under water stress via involvement in starch metabolism, antioxidant defenses and relevant gene expression. Molecules 19:18003–18024

    Article  PubMed  Google Scholar 

  • Li Z, Peng Y, Zhang XQ, Pan MH, Ma X, Huang LK, Yan YH (2014b) Exogenous spermidine improves water stress tolerance of white clover (Trifolium repens L.) involved in antioxidant defence, gene expression and proline metabolism. Plant Omics 7:517–526

    CAS  Google Scholar 

  • Li Z, Zhou H, Peng Y, Zhang XQ, Ma X, Huang LK, Yan YH (2015) Exogenously applied spermidine improves drought tolerance in creeping bentgrass associated with changes in antioxidant defense, endogenous polyamines and phytohormones. Plant Growth Regul 76:71–82

    Article  CAS  Google Scholar 

  • Liu JH, Kitashiba H, Wang J, Ban Y, Moriguchi T (2007) Polyamines and their ability to provide environmental stress tolerance to plants. Plant Biotechnol J 24:117–126

    Article  CAS  Google Scholar 

  • Liu J, Liu GH, Hou LX, Liu X (2010) Ethylene-induced nitric oxide production and stomatal closure in Arabidopsis thaliana depending on changes in cytosolic pH. Chin Sci Bull 55:2403–2409

    Article  CAS  Google Scholar 

  • Lu S, Su W, Li H, Guo Z (2009) Abscisic acid improves drought tolerance of triploid bermudagrass and involves H2O2-and NO-induced antioxidant enzyme activities. Plant Physiol Biochem 47:132–138

    Article  CAS  PubMed  Google Scholar 

  • Martin-Tanguy J (2001) Metabolism and function of polyamines in plants: recent development (new approaches). Plant Growth Regul 34:135–148

    Article  CAS  Google Scholar 

  • Miller G, Suzuki N, Ciftci-Yilmaz S, Mittler R (2010) Reactive oxygen species homeostasis and signalling during drought and salinity stresses. Plant Cell Environ 33:453–467

    Article  CAS  PubMed  Google Scholar 

  • Mostofa MG, Yoshida N, Fujita M (2014) Spermidine pretreatment enhances heat tolerance in rice seedlings through modulating antioxidative and glyoxalase systems. Plant Growth Regul 73:31–44

    Article  CAS  Google Scholar 

  • Nakano Y, Asada K (1981) Hydrogen peroxide is scavenged by ascorbate-specific peroxidase in spinach chloroplasts. Plant Cell Physiol 22:867–880

    CAS  Google Scholar 

  • Parvin S, Lee OR, Sathiyaraj G, Khorolragchaa A, Kim YJ, Yang DC (2014) Spermidine alleviates the growth of saline-stressed ginseng seedlings through antioxidative defense system. Gene 537:70–78

    Article  CAS  PubMed  Google Scholar 

  • Radhakrishnan R, Lee IJ (2013) Ameliorative effects of spermine against osmotic stress through antioxidants and abscisic acid changes in soybean pods and seeds. Acta Physiol Plant 35:263–269

    Article  CAS  Google Scholar 

  • Sang J, Jiang M, Lin F, Xu S, Zhang A, Tan M (2008) Nitric oxide reduces hydrogen peroxide accumulation involved in water stress-induced subcellular anti-oxidant defense in maize plants. J Integr Plant Biol 50:231–243

    Article  CAS  PubMed  Google Scholar 

  • Shi H, Chan Z (2014) Improvement of plant abiotic stress tolerance through modulation of the polyamine pathway. J Integr Plant Biol 56:114–121

    Article  CAS  PubMed  Google Scholar 

  • Shi J, Fu XZ, Peng T, Huang XS, Fan QJ, Liu JH (2010) Spermine pretreatment confers dehydration tolerance of citrus in vitro plants via modulation of antioxidative capacity and stomatal response. Tree Physiol 30:914–922

    Article  CAS  PubMed  Google Scholar 

  • Shi HT, Li RJ, Cai W, Liu W, Fu ZW, Lu YT (2012) In vivo role of nitric oxide in plant response to abiotic and biotic stress. Plant Signal Behav 7:437–439

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Shi H, Ye T, Chan Z (2013a) Comparative proteomic and physiological analyses reveal the protective effect of exogenous polyamines in the bermudagrass (Cynodon dactylon) response to salt and drought stresses. J Proteome Res 12:4951–4964

    Article  CAS  PubMed  Google Scholar 

  • Shi H, Ye T, Chen F, Cheng Z, Wang Y, Yang P, Chan Z (2013b) Manipulation of arginase expression modulates abiotic stress tolerance in Arabidopsis: effect on arginine metabolism and ROS accumulation. J Exp Bot 64:1367–1379

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Shi H, Ye T, Zhu JK, Chan Z (2014) Constitutive production of nitric oxide leads to enhanced drought stress resistance and extensive transcriptional reprogramming in Arabidopsis. J Exp Bot 65:4119–4131

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Shukla V, Ma YM, Merewitz E (2015) Creeping bentgrass responses to drought stress and polyamine application. J Am Soc Hort Sci 140:94–101

    CAS  Google Scholar 

  • Siddiqui MH, Al-Whaibi MH, Basalah MO (2011) Role of nitric oxide in tolerance of plants to abiotic stress. Protoplasma 248:447–455

    Article  CAS  PubMed  Google Scholar 

  • Sobieszczuk-Nowicka E, Rorat T, Legocka J (2007) Polyamine metabolism and S-adenosylmethionine decarboxylase gene expression during the cytokinin-stimulated greening process. Acta Physiol Plant 29:495–502

    Article  CAS  Google Scholar 

  • Steiner N, Santa-Catarina C, Silveira V, Floh EI, Guerra MP (2007) Polyamine effects on growth and endogenous hormones levels in Araucaria angustiolia embryogenic cultures. Plant Cell Tissue Organ Cult 89:55–62

    Article  CAS  Google Scholar 

  • Tian J, Wang LP, Yang YJ, Sun J, Guo SR (2012) Exogenous spermidine alleviates the oxidative damage in cucumber seedlings subjected to high temperatures. J Am Soc Hort Sci 137:11–19

    Article  CAS  Google Scholar 

  • Tun NN, Santa-Catarina C, Begum T, Silveira V, Handro W, Floh EI, Scherer GF (2006) Polyamines induce rapid biosynthesis of nitric oxide (NO) in Arabidopsis thaliana seedlings. Plant Cell Physiol 47:346–354

    Article  CAS  PubMed  Google Scholar 

  • Wang HH, Huang JJ, Bi YR (2010) Nitrate reductase-dependent nitric oxide production is involved in aluminum tolerance in red kidney bean roots. Plant Sci 179:281–288

    Article  CAS  Google Scholar 

  • Wimalasekera R, Tebartz F, Scherer GF (2011) Polyamines, polyamine oxidases and nitric oxide in development, abiotic and biotic stresses. Plant Sci 181:593–603

    Article  CAS  PubMed  Google Scholar 

  • Xia XJ, Wang XJ, Zhou YH, Tao Y, Mao WH, Shi K, Yu JQ (2009) Reactive oxygen species are involved in brassinosteroid-induced stress tolerance in cucumber. Plant Physiol 150:801–814

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Yamamoto A, Shim IS, Fujihara S (2012) Chilling-stress responses by rice seedlings grown with different ammonium concentrations and its relationship to leaf spermidine content. J Plant Biol 55:191–197

    Article  CAS  Google Scholar 

  • Yang JC, Zhang JH, Liu K, Wang ZQ, Liu LJ (2007) Involvement of polyamines in the drought resistance of rice. J Exp Bot 58:1545–1555

    Article  CAS  PubMed  Google Scholar 

  • Yang BN, Wu JZ, Gao FM, Wang J, Su GX (2014) Polyamine-induced nitric oxide generation and its potential requirement for peroxide in suspension cells of soybean cotyledon node callus. Plant Physiol Biochem 79:41–47

    Article  PubMed  Google Scholar 

  • Yin ZP, Li S, Ren J, Song XS (2014) Role of spermidine and spermine in alleviation of drought-induced oxidative stress and photosynthetic inhibition in Chinese dwarf cherry (Cerasus humilis) seedlings. Plant Growth Regul 74:209–218

    Article  CAS  Google Scholar 

  • Zhang Y, Wang L, Liu Y, Zhang Q, Wei Q, Zhang W (2006) Nitric oxide enhances salt tolerance in maize seedlings through increasing activities of proton-pump and Na+/H+ antiport in the tonoplast. Planta 224:545–555

    Article  CAS  PubMed  Google Scholar 

  • Zhang A, Zhang J, Zhang J, Ye N, Zhang H, Tan M, Jiang M (2011) Nitric oxide mediates brassinosteroid-induced ABA biosynthesis involved in oxidative stress tolerance in maize leaves. Plant Cell Physiol 52:181–192

    Article  CAS  PubMed  Google Scholar 

  • Zhao MG, Chen L, Zhang LL, Zhang WH (2009) Nitric reductase-dependent nitric oxide production is involved in cold acclimation and freezing tolerance in Arabidopsis. Plant Physiol 151:755–767

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Zhou B, Guo Z, Xing J, Huang B (2005) Nitric oxide is involved in abscisic acid-induced antioxidant activities in Stylosanthes guianensis. J Exp Bot 56:3223–3228

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

Financial support was obtained from the Natural Science Foundation of China (Grant No. 31372371), National Support Program (Grant No. 2011BAD17B03), and Sichuan Province Support Program (Grant No. 2013NZ0013).

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The authors declare that they have no competing interests.

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    Authors and Affiliations

    1. Key Laboratory of Grassland Science, Faculty of Animal Science and Technology, Sichuan Agricultural University, Huiming Street 211, Chengdu, Sichuan, 611130, People’s Republic of China

      Dandan Peng, Xiaojuan Wang, Zhou Li, Yan Zhang, Yan Peng, Yaping Li, Xiaoshuang He, Xinquan Zhang, Xiao Ma, Linkai Huang & Yanhong Yan

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    1. Dandan Peng
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    2. Xiaojuan Wang
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    Correspondence to Yan Peng.

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    Handling Editor: Bhumi Nath Tripathi

    Dandan Peng and Xiaojuan Wang contributed equally to this work.

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    Peng, D., Wang, X., Li, Z. et al. NO is involved in spermidine-induced drought tolerance in white clover via activation of antioxidant enzymes and genes. Protoplasma 253, 1243–1254 (2016). https://doi.org/10.1007/s00709-015-0880-8

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