Combined hydrogen peroxide and nitric oxide priming modulate salt stress tolerance in acclimated and non-acclimated oilseed rape (Brassica napus L.) plants

Document Type : Research Paper


1 Department of Biology, Faculty of Sciences, Urmia University, Urmia, Iran.

2 Department of Biology, Faculty of Science, Urmia University, Urmia, Iran.

3 Department of Biology, Payame Noor University (PNU), Tehran, Iran.



We examined the combined effects of hydrogen peroxide (H2O2) and nitric oxide (NO) on the responses of oilseed rape(Brassica napus L.) plants to salt stress under acclimated and non-acclimated conditions. The results of the shoot and root dry weight traits together with the measurement of malondialdehyde (MDA) indicated that salt acclimation with a low concentration of NaCl (50 mM) could not alleviate the inhibitory effect of high salinity (200 mM NaCl). Under acclimated conditions, seed priming with H2O2 or NO resulted in effective protection against salt stress, however, maximum amelioration of salt stress was found by the combined treatments of H2O2 + NO. Interestingly, in the salt-exposed non-acclimated plants, only seed priming with H2O2 + NO was effective in improving salt tolerance. Pretreatment with H2O2 + NO tended to limit Na translocation into photosynthetic organs to prevent salt damages. Additionally, a large increase in salicylic acid contentwas correlated with phenylalanine ammonia lyase activation and flavonoid biosynthesis was observed when oilseed rape plants exposed to salinity in the presence of H2O2+NO. Interestingly, in this study, endogenous NO content of H2O2–primed plants exhibited a significant increase under non-saline conditions, indicating that H2O2 influences NO accumulation. In addition, oilseed rape plants primed with H2O2 + NO exhibited lower MDA and H2O2 content, contributing to the better induction of antioxidative enzyme activities. Higher levels of antioxidant enzyme activities maintained the integrity of cell membranes, resulting in better plant growth under salt stress. Taken together, our results revealed that oilseed rape plants pretreated with H2O2 + NO exhibited more effective tolerance to salt stress than plants that were pretreated with H2O2 or NO alone.


Article Title [فارسی]

بهبود تحمل به شوری در گیاه کلزا توسط پرایمینگ ترکیبی پراکسید هیدروژن و اکسید نیتریک در شرایط عادت دهی و بدون عادت دهی به شوری

Authors [فارسی]

  • زهرا کریمی 1
  • جلیل خارا 2
  • قادر حبیبی 3
1 گروه زیست شناسی، دانشکده علوم، دانشگاه ارومیه، ارومیه.
2 گروه زیست شناسی، دانشکده علوم، دانشگاه ارومیه، ارومیه.
3 گروه زیست شناسی، دانشگاه پیام نور، تهران.
Abstract [فارسی]

در این پژوهش، تأثیر پرایمینگ بذور با پراکسید هیدروژن (H2O2)، اکسید نیتریک (NO) یا ترکیب H2O2+NO بر پاسخ های گیاه کلزا به شوری در شرایط عادت­ دهی و  بدون عادت­ دهی به شوری مورد مطالعه قرار گرفت. نتایج حاصل از سنجش صفات وزن خشک ساقه و ریشه به همراه سنجش مالون دی آلدئید (MDA) نشان دادند که کاربرد غلظت اندک کلرید سدیم (50 میلی مولار) به عنوان عامل عادت­ دهی به شوری، نتوانست باعث تخفیف اثرات زیانبار شوری بالا (200 میلی مولار) شود. در عین حال، در شرایط عادت­ دهی، پرایمینگ بذور با H2O2یا NO باعث تخفیف اثرات تنش شوری بر رشد کلزا شد، هرچند بیشترین تخفیف توسط ترکیب H2O2+NO به دست آمد. جالب توجه این که در گیاهان بدون عادت­ دهی به شوری، تنها پرایمینگ ترکیبی بذور با H2O2+NO در بهبود تحمل به شوری موثر بود. این تخفیف تنش توسط پرایمینگ ترکیبی در شرایط شوری، با کاهش انتقال سدیم به اندام هوایی، افزایش مقدار اسید سالیسیلیک، فلاوونوئیدها و فعالیت آنزیم فنیل آلانین آمونیالیاز همراه بود. پرایمینگ بذور با پراکسید هیدروژن در شرایط غیر­شور، باعث افزایش مقدار NO داخلی شد. همچنین، پرایمینگ بذور با ترکیب H2O2+NO از طریق کاهش MDA و H2O2داخلی وافزایش فعالیت آنزیم­ های سیستم آنتی اکسیدان، باعث حفظ تمامیت غشاها و در نتیجه بهبود رشد در شرایط شوری گردید. نتایج این تحقیق نشان داد که پرایمینگ ترکیبی بهتر از پرایمینگ تنها توانست اثرات زیانبار شوری در گیاه کلزا را تخفیف دهد.

Keywords [فارسی]

  • اسید سالیسیلیک
  • اکسید نیتریک
  • پرایمینگ ترکیبی
  • شوری
  • فلاوونوئید
  • کلزا
  • هومئوستازی یونی
Ali Q, Daud MK, Haider MZ, Ali S, Rizwan M, Aslam N, Noman A, Iqbal N, Shahzad F, Deeba F and Ali I, 2017. Seed priming by sodium nitroprusside improves salt tolerance in wheat (Triticum aestivum L.) by enhancing physiological and biochemical parameters. Plant Physiology and Biochemistry 119: 50-58.
Ashfaque F, Khan MIR and Khan NA, 2014. Exogenously applied H2O2 promotes proline accumulation, water relations, photosynthetic efficiency and growth of wheat (Triticum aestivum L.) under salt stress. Annual Research & Review in Biology 4(1):105-120.
Balotf S, Islam S, Kavoosi G, Kholdebarin B, Juhasz A and Ma W, 2018. How exogenous nitric oxide regulates nitrogen assimilation in wheat seedlings under different nitrogen sources and levels. PLoS One 13(1):e0190269. 10.1371/journal.pone.0190269.
Bates LS, Waldren RP and Teare ID, 1973. Rapid determination of free proline for water-stress studies. Plant and Soil 39(1): 205-207.
Blasco B, Leyva R, Romero L and Ruiz JM, 2013. Iodine effects on phenolic metabolism in lettuce plants under salt stress. Journal of Agricultural and Food Chemistry 61: 2591-2596.
Bradford MM, 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72 (1-2): 248-254.
Burchard P, Bilger W and Weissenbock G, 2000. Contribution of hydroxycinnamates and flavonoids to epidermal shielding of UV‐A and UV‐B radiation in developing rye primary leaves as assessed by ultraviolet‐induced chlorophyll fluorescence measurements. Plant, Cell and Environment 23: 1373-1380.
Chun SC, Paramasivan M and Chandrasekaran M, 2018. Proline accumulation influenced by osmotic stress in Arbuscular mycorrhizal symbiotic plants.Frontiers in Microbiology9:2525. doi: 10.3389/fmicb.2018.02525.
David A, Yadav S and Bhatla SC, 2010. Sodium chloride stress induces nitric oxide accumulation in root tips and oil body surface accompanying slower oleosin degradation in sunflower seedlings. Physiologia Plantarum 140: 342-354.
Egbichi I, Keyster M and Ludidi N, 2014. Effect of exogenous application of nitric oxide on salt stress responses of soybean. South African Journal of Botany 90: 131-136.
Ellouzi H, Sghayar S and Abdelly C, 2017. H2O2 seed priming improves tolerance to salinity; drought and their combined effect more than mannitol in Cakile maritima when compared to Eutrema salsugineum. Journal of Plant Physiology 210: 38-50.
Farzane A, Nemati H, Shoor M and Ansari H, 2020. Antioxidant enzyme and plant productivity changes in field-grown tomato under drought stress conditions using exogenous putrescine. Journal of Plant Physiology and Breeding 10: 29-40.
Geranpayeh A, Azizpour K, Vojodi Mehrabani L and Valizadeh Kamran R, 2017. Effects of salinity on some physiological characteristics of Lepidium sativum L. Journal of Plant Physiology and Breeding 7: 23-30.
Gondim FA, Gomes-Filho E, Costa JH, Mendes Alencar NL and Prisco JT, 2012. Catalase plays a key role in salt stress acclimation induced by hydrogen peroxide pretreatment in maize. Plant Physiology and Biochemistry 56: 62-71.
Gondim FA, Miranda RD, Gomes-Filho E and Prisco JT, 2013. Enhanced salt tolerance in maize plants induced by H2O2 leaf spraying is associated with improved gas exchange rather than with non-enzymatic antioxidant system. Theoretical and Experimental Plant Physiology 25(4): 251-60.
Guo Z, Tan J, Zhuo C, Wang C, Xiang B and Wang Z, 2014. Abscisic acid, H2O2 and nitric oxide interactions mediated cold-induced S-adenosylmethionine synthetase in Medicago sativa subsp. falcata that confers cold tolerance through up regulating polyamine oxidation. Plant Biotechnology Journal 12(5): 601-612.
Habibi G, 2017. Physiological, photochemical and ionic responses of sunflower seedlings to exogenous selenium supply under salt stress. Acta Physiologiae Plantarum 39(10): 213. doi:10.1007/s11738-017-2517-3.
Habibi G, 2019. Role of exogenous hydrogen peroxide and nitric oxide on improvement of abiotic stress tolerance in plants. In: Hasanuzzaman M, Fujita M, Oku H and Islam MT (eds). Plant Tolerance to Environmental Stress: Role of Phytoprotectants, pp. 159-174. CRC Press.
Habibi G and Hajiboland R, 2012. Comparison of photosynthesis and antioxidative protection in Sedum album and Sedum stoloniferum (Crassulaceae) under water stress. Photosynthetica 50: 508-518.
Hajiboland R, Aliasgharzadeh N, Laiegh SF and Poschenrieder C, 2010. Colonization with arbuscular mycorrhizal fungi improves salinity tolerance of tomato (Solanum lycopersicum L.) plants. Plant and Soil 331: 313-327.
Hameed A, HussainT, Gulzar, Aziz F, Gul B and Khan MA, 2012. Salt tolerance of a cash crop halophyte Suaeda fruticosa: biochemical responses to salt and exogenous chemical treatments. Acta Physiologiae Plantarum 34: 2331-2340.
Hayat S, Yadav S, Wani AS, Irfan M, Alyemini MN and Ahmad A, 2012. Impact of sodium nitroprusside on nitrate reductase, proline content, and antioxidant system in tomato under salinity stress. Horticulture, Environment, and Biotechnology 53: 362-367.
Huang AX, Wang YS, She XP, Mu J and Zhao JL, 2015. Copper amine oxidase-catalysed hydrogen peroxide involves production of nitric oxide in darkness-induced stomatal closure in broad bean. Functional Plant Biology 42(11):1057-1067.
Islam MM, Hoque MA, Okuma E, Banu MN, Shimoishi Y, Nakamura Y and Murata Y, 2009. Exogenous proline and glycinebetaine increase antioxidant enzyme activities and confer tolerance to cadmium stress in cultured tobacco cells. Journal of Plant Physiology 166: 1587-1597.
Jiang C, Zu C, Lu D, Zheng Q, Shen J, Wang H and Li D, 2017. Effect of exogenous selenium supply on photosynthesis, Na+ accumulation and antioxidative capacity of maize (Zea mays L.) under salinity stress. Scientific Reports 7: 42039. doi: 10.1038/srep42039.
Kadioglu A, Saruhan N, Saglam A, Terzi, R and Acet T, 2011. Exogenous salicylic acid alleviates effects of long term drought stress and delays leaf rolling by inducing antioxidant system. Plant Growth Regulation 64: 27-37.
Khan MN, Siddiqui MH, Mohamma DF and Naeem M, 2012. Interactive role of nitric oxide and calcium chloride in enhancing tolerance to salt stress. Nitric Oxide 27(4): 210-218.
Kholghi M, Toorchi M, Bandeh-Hagh A and Shakiba MR, 2018. An evaluation of oilseed rape genotypes under salinity stress at vegetative stage via morphological and physiological traits. Pakistan Journal of Botany 50(2): 447-455.
Kilic S and Kahraman A, 2016. The mitigation effects of exogenous hydrogen peroxide when alleviating seed germination and seedling growth inhibition on salinity-induced stress in barley. Polish Journal of Environmental Studies 25(3): 1053-1059.
Li T, Hu Y, Du X, Tang H, Shen C and Wu J, 2014. Salicylic acid alleviates the adverse effects of salt stress in Torreya grandis cv. merrillii seedlings by activating photosynthesis and enhancing antioxidant systems. PLoS ONE 9(10): e109492.
Li JT, Qiu ZB, Zhang XW and Wang LS, 2011. Exogenous hydrogen peroxide can enhance tolerance of wheat seedlings to salt stress. Acta Physiologiae Plantarum 33: 835-842.
Li X, Zhang L, Ahamed GJ, Li YT, Wei JP, Yan P, Zhang LP, Han X and Han WY, 2019. Salicylic acid acts upstream of nitric oxide in elevated carbon dioxide-induced flavonoid biosynthesis in tea plant (Camellia sinensis L.). Environmental and Experimental Botany 161: 367-374.
Li ZG, Luo LJ and Sun YF, 2015. Signal crosstalk between nitric oxide and hydrogen sulfide may be involved in hydrogen peroxide–induced thermotolerance in maize seedlings. Russian Journal of Plant Physiology 62: 507-514.
Li ZG, Yang SZ, Long WB, Yang GX and Shen ZZ, 2013. Hydrogen sulphide may be a novel downstream signal molecule in nitric oxide‐induced heat tolerance of maize (Zea mays L.) seedlings. Plant, Cell & Environment 36(8): 1564-1572.
Ma X, Ou YB, Gao YF, Lutts S, Li TT, Wang Y, Chen YF, Sun YF and Yao YA, 2016. Moderate salt treatment alleviates ultraviolet-B radiation caused impairment in poplar plants. Scientific Reports 6: 32890.
Magne C, Saladin G and Clement C, 2006. Transient effect of the herbicide flazasulfuron on carbohydrate physiology in Vitis vinifera. Chemosphere 62(4): 650-657.
Mahmoudi H, Huang J, Gruber MY, Kaddour R, Lachaal M, Ouerghi Z and Hannoufa A, 2010. The impact of genotype and salinity on physiological function, secondary metabolite accumulation, and antioxidative responses in lettuce. Journal of Agricultural and Food Chemistry 58(5): 5122-2130.
Manai J, Gouia H and Corpas FJ, 2014. Redox and nitric oxide homeostasis are affected in tomato (Solanum lycopersicum) roots under salinity-induced oxidative stress. Journal of Plant Physiology 171(12): 1028-1035.
Meda A, Lamien CE, Romito M, Millogo J and Nacoulma OG, 2005. Determination of the total phenolic, flavonoid and proline contents in Burkina Fasan honey, as well as their radical scavenging activity. Food Chemistry 91: 571-577.
Miller GA, Suzuki N, Ciftci-Yilmaz, S and Mittler R, 2010. Reactive oxygen species homeostasis and signalling during drought and salinity stresses. Plant, Cell and Environment 33(4): 453-467.
Munns R and Tester M, 2008. Mechanisms of salinity tolerance. Annual Review of Plant Biology 59:651-681.
Niu L and Liao W, 2016. Hydrogen peroxide signaling in plant development and abiotic responses: crosstalk with nitric oxide and calcium. Frontiers in Plant Science 7: 230.
Oh MM, Trick HN and Rajashekar CB, 2009. Secondary metabolism and antioxidants are involved in environmental adaptation and stress tolerance in lettuce. Journal of Plant Biology 166(2): 180-191.
Pandolfi C, Mancusoa S. and Shabalab S. 2012. Physiology of acclimation to salinity stress in pea (Pisum sativum). Environmental and Experimental Botany 84: 44-51.
Parida AK and Das AB, 2005. Salt tolerance and salinity effects on plants: a review. Ecotoxicology and Environmental Safety 60(3): 324-349.
Quentin AG, Pinkard EA, Ryan MG, Tissue DT, Baggett LS, Adams HD, Maillard P, Marchand J, Landhäusser SM, Lacointe A and Gibon Y, 2015. Non-structural carbohydrates in woody plants compared among laboratories. Tree Physiology 35(11): 1146-1165.
Rajabi S, Karimzadeh G and Ghanati F, 2012. Salt-induced changes of antioxidant enzymes activity in winter canola (Brassica napus) cultivars in growth chamber. Jounal of Plant Physiology and Breeding 2: 11-21.
Sathiyaraj G, Srinivasan S, Kim Y.J, Lee OR, Balusamy SDR and Khorolaragchaa A, 2014. Acclimation of hydrogen peroxide enhances salt tolerance by activating defense-related proteins in Panax ginseng CA. Meyer. Molecular Biology Reports 41: 3761-3771.
Savvides A, Ali S, Tester M and Fotopoulos V, 2016. Chemical priming of plants against multiple abiotic stresses: mission possible? Trends in Plant Science 21(4): 329-340.
Shi K, Li X, Zhang H, Zhang G, Liu Y, Zhou Y, Xia X, Chen Z and Yu J, 2015. Guard cell hydrogen peroxide and nitric oxide mediate elevated CO2‐induced stomatal movement in tomato. New Phytologist 208(2): 342-353.
Su H, Song S, Yan X, Fang L, Zeng B and Zhu Y, 2018. Endogenous salicylic acid shows different correlation with baicalin and baicalein in the medicinal plant Scutellaria baicalensis Georgi subjected to stress and exogenous salicylic acid. PLoS One 13(2): e0192114.
Tan J, Wang C, Xiang B, Han R and Guo Z, 2013. Hydrogen peroxide and nitric oxide mediated cold- and dehydration-induced myo-inositol phosphate synthase that confers multiple resistances to abiotic stresses. Plant, Cell and Environment 36(2): 288-299.
Tanou G, Filippou P, Belghazi M, Job D, Diamantidis G, Fotopoulos V and Molassiotis A, 2012. Oxidative and nitrosative‐based signaling and associated post‐translational modifications orchestrate the acclimation of citrus plants to salinity stress. The Plant Journal 72(4): 585-599.
Tanou G, Job C, Rajjou L, Arc E, Belghazi M and Diamantidis G, 2009. Proteomics reveals the overlapping roles of hydrogen peroxide and nitric oxide in the acclimation of citrus plants to salinity. The Plant Journal 60: 795-804.
Terzi R, Kadioglu A, Kalaycioglu E and Saglam A, 2014. Hydrogen peroxide pretreatment induces osmotic stress tolerance by influencing osmolyte and abscisic acid levels in maize leaves. Journal of Plant Interactions 9: 559-565.
Tossi V, Lamattina L, Jenkins GI and Cassia RO, 2014. Ultraviolet-B-induced stomatal closure in Arabidopsis is regulated by the UV Resistance Locus and photoreceptor in a nitric oxide-dependent mechanism. Plant Physiology 164(4): 2220-2230.
Velikova V, Yordanov I and Edreva A, 2000. Oxidative stress and some antioxidant systems in acid rain-treated bean plants: protective role of exogenous polyamines. Plant Science 151(1): 59-66.
Velioglu YS, Mazza G, Gao L and Oomah BD, 1998. Antioxidant activity and total phenolics in selected fruits, vegetables, and grain products. Journal of Agricultural and Food Chemistry 46: 4113-4117.
Wang L, Guo Y, Jia L, Chu H, Zhou S, Chen K, Wu D and Zhao L, 2014. Hydrogen peroxide acts upstream of nitric oxide in the heat shock pathway in Arabidopsis seedlings. Plant Physiology 164(4): 2184-2196.
Wu Q, Su N, Zhang X, Liu Y, Cui J and Liang Y, 2016. Hydrogen peroxide, nitric oxide and UV Resistance Locus and interact to mediate UV-B-induced anthocyanin biosynthesis in radish sprouts. Scientific Reports 6: 29164.
Xu Z and Rothstein SJ, 2018. ROS-induced anthocyanin production provides feedback protection by scavenging ROS and maintaining photosynthetic capacity in Arabidopsis. Plant Signaling & Behavior 13(3): e1451708. doi: 10.1080/15592324.2018.1451708.
Yang L, Zhao X, Zhu H, Paul M, Zu Y and Tang Z, 2014. Exogenous trehalose largely alleviates ionic unbalance, Ros burst, and Pcd occurrence induced by high salinity in Arabidopsis seedlings. Frontiers in Plant Science 5: 570. doi: 10.3389/fpls.2014.00570.
Zhang M, Tang S, Huang X, Zhang F, Pang Y, Huang Q and Yi Q, 2014. Selenium uptake, dynamic changes in selenium content and its influence on photosynthesis and chlorophyll fluorescence in rice (Oryza sativa L.). Environmental and Experimental Botany107: 39-45.
Zucker M, 1965. Induction of phenylalanine deaminase by light and its relation to chlorogenic acid synthesis in potato tuber tissue. Plant Physiology 40(5): 779-784.