Effect of Nitric Oxide and Arbuscular Mycorrhiza on some Physiological Traits of Liquorice (Glycyrrhiza glabra L.) Plant under Salinity Stress

Document Type : Research Article


1 Islamic Azad University of Islamshahr, Tehran

2 Shahed University, Tehran


Introduction: The salinity affliction of land constitutes a major threat amongst the various forms of soil degradation. Arbuscular mycorrhiza fungus can be useful as a bio-fertilizer in providing plant nutrition and reducing the effects of environmental stresses on plants. On the other hand, nitric oxide plays a role in many environmental and non-environmental stresses, including drought and salinity stresses. Liquorice (Glycyrrhiza globra Linn.), commonly known as Mulahatti and Yashtimadhu, is the highest priority value crop which can be successfully cultivated on salt-affected and degraded lands. It is a small perennial leguminous herb of the family Fabaceae (Papilionaceae) native to the Mediterranean region and central and southwest Asia, and cultivated in Italy, Russia, France, UK, USA, Germany, Spain, China, Pakistan, Afghanistan, Iran, Iraq, Uzbekistan, Turkey, Turkmenistan and north-western India. This research was carried out with the aim of investigating the effect of nitric oxide modification on coexistence with arbuscular mycorrhizal fungus on some of the physiological traits of licorice under the salt stress of sodium chloride.
Materials and Methods: This research was a factorial experiment based on completely randomized block design with three replications. Factors consisted of five levels of NaCl-salinity (0 as control, 50, 100, 150 and 200 mM), two levels of nitric oxide (0 and 0.2 mM) and two levels of mycorrhizal fungi (the presence and absence of mycorrhizal). To do this, 10 kg pot of pumice mixture and pumice (1 to 1 ratio) were poured into 60 plastic containers (30 x 20 cm; 10 L) and sterilized by alcohol. The seeds germinated in petri dishes after adequate growth, they were transferred to the pots (all seeds were germinated and grown in the same conditions). In each pot, five seedlings were cultured and irrigated with distilled water until a two-leaf stage. After that, the treatment was carried out by a Hoagland solution. Application of saline treatments and nitric oxide (from sodium nitroproced as nitric oxide source) was performed 45-days. Finally, after 60 days of planting, sampling was carried out to measure the physiological traits from the middle leaves of each pot, and after being placed in an aluminum foil with ice-containing flux, it was transferred to the laboratory and then transferred to 80 o C. The evaluated traits were leaf flavonoids by Swain (52) method, proline content by Bates et al. (6) method, MDA with Ohkawa et al. (40) method, CAT activity by Pereira et al. (44) method, POD activity by Korori (28) method and SOD activity by Giannopolitis and Reis (21) method. The data were analyzed by SPSS 22 (IBM SPSS Statistics 22.0) software application. The data was normalized and inferential statistics such as analysis of variance and mean comparison of treatments were calculated using Duncan's multiple range test.
Results and Discussion: The results showed that the salinity stress had significant effect on flavonoid content, proline content, malondialdehyde rate and antioxidant activity of catalase, peroxidase and superoxide dismutase. Salinity had increased levels of malondialdehyde, proline content, and the activity of antioxidant enzymes (catalase, peroxidase, and superoxide dismutase). The coexistence of mycorrhiza fungus in combination with nitric oxide or alone reduced the number of flavonoids and increased proline content at each level of salinity stress. Nitric oxide had no significant effect on measured traits but was more effective in combination with Mycorrhiza fungi. In general, sodium chloride salinity stress had a negative effect on the physiological traits of liquorice, but the use of nitric oxide with arbuscular mycorrhizal fungus reduced the negative effects of stress. In general, it can be said that the removal and decontamination of active oxygen species is an important part of salinity tolerance in plants. In the present study, salinity stresses have significantly increased the amount of MDA, which is an indicator of plant response to stress. In addition to salinity stress, nitric oxide stress has been induced to reduce the effects of high salt concentration on some of the indices, thus reducing nitric oxide in high concentrations of MDA. Application of saline treatment significantly increased the activity of the three antioxidant enzymes CAT, POD, and SOD. The results showed that salinity stress had a decreasing effect on studied traits, but the application of arbuscular mycorrhizal fungus with nitric oxide reduced the negative effects of sodium chloride salinity stress on liquorice plant.


1- Agyare, C., Boakye, Y.D., Bekoe, E.O., Hensel, A., Dapaah, S.O., and Appiah, T. 2015. Review: African medicinal plants with wound healing properties. Journal of ethnopharmacology, 65(4): 245-255.
2- Akram, M.S., and Ashraf, M. 2009. Alleviation of adverse effects of salt stress on sunflower (Helianthus annuus L.) by exogenous application of potassium nitrate. Journal of Applied Botany and Food Quality, 83:19-27.
3- Amirjani, M.R. 2010. Effect of NaCl on some physiological parameters of rice. European Journal of Biological Sciences, 3 (1): 06-16.
4- Appel, K., and Hirt, H. 2004. Reactive oxygen species: metabolism, oxidative stress and signal transduction. Annual Review Plant Biology, 55: 373-399.
5- Ashraf, M., Orooj A. 2006. Salt stress effects on growth, ion accumulation and seed oil concentration in arid zone traditional medicinal plant ajwain (Trachyspermum ammi [L.] Sprague). Journal of Arid Environment, 64: 209-220.
6- Bates, L., Waldren, R.P., and Teare, I.D. 1973. Rapid determination of free proline for water-stress studies. Plant and Soil, 39: 205-207.
7- Beligni, M.V., Fath, A., Bethke, P.C., Lamattina, L., and Jones, R.L. 2002. Nitric oxideacts as an antioxidant and delays programmed cell death in barley aleurone layers. Plant Physiology, 129: 1642-1650.
8- Bor, M., Özdemir, F., and Türkan, I. 2003. The effect of salt stress on lipid peroxidation and antioxidants in leaves of sugar beet Beta vulgaris L. and wild beet Beta maritima L. Plant Science Journal, 164: 77–84.
9- Borsani, O., Valpuesta, V., and Botella, M.A. 2001. Evidence for a role of salicylic acid in the oxidative damage generated by NaCl and osmotic stress in Arabidopsis seedlings. Plant Physiology, 126: 1024–1030.
10- Brayant, J.P., Chapin, F.S., and Klein D.R. 1983. Carbon nutrient balance of boreal plants in relation to vertebrate herbivory. Oikos Journal, 40: 357-368.
11- Butler, A.R., and Megson, I.L. 2002. Non-heme iron nitrosyls in biology. Chemical Review Journal, 102(4): 1155-1165.
12- Chaparzadeh, N., D'Amico, M.L., Khavari-Nejad, R.A., Izzo, R., and Navari-Izzo, F. 2004. Antioxidative responses of Calendula officinalis L. under salinity conditions. Plant Physiology and Biochemistry, 42: 695-701.
13- Chaparzadeh, N., Pajang, M., and Mohammadpour, A. 2015. The role of nitric oxide precursor in antioxidant responses of chickpea when reducing the nightly temperature. Process and Plant Function, 12 (4): 9-1.
14- Chohan, M., Naughton, D.P., Jones, L., and Opara, E.I. 2012. An investigation of the relationship between the anti-inflammatory activity, polyphenolic content and antioxidant activities of cooked and in vitro digested culinary herbs. Oxidative Medicine and Cellular Longevity, P: 1-9. doi:10.1155/2012/627843.
15- Cinatl, J., Morgenstern, B., Bauer, G., Chandra, P., Rabenau, H., and Doerr, H.W. 2003. Glycyrrhizin, an active component of liquorice roots, and replication of SARS-associated coronavirus. The Lancet Medical Journal, 361: 2045– 2046.
16- Dat, J., Vandenabeele, S., Vranova, E., Van Montagu, M., Inze, D., and Van Breusegem, F. 2000. Dual action of the active oxygen species during plant stress responses. Cellular and Molecular Life Sciences, 57: 779-795.
17- Del Rio, L.A., Sandalio, L.M., Corpas, F.J., Palma, J.M., and Barroso, J.B. 2006. Reactive oxygen species and reactive nitrogen species in peroxisomes production, scavenging, and role in cell signaling. Plant Physiology, 141: 330–335.
18- Demiral, T., and Turkan, I. 2005. Comparative lipid peroxidation, antioxidant defense systems and proline content in roots of two rice cultivars differing in salt tolerance. Environmental Experiment Botany, 53: 247–257.
19- Duan, X., Jiang, Y., Su, X., Zhang, Z., and Shi, J. 2007. Antioxidant properties of anthocyanins extracted from litchi (Litchi chinenesis Sonn.) fruit pericarp tissues in relation to their role in the pericarp browning. Food Chemistry, 101:1365–1371.
20- Esmaeilzadeh Bahabadi, S., Rezaei Nodehi, A., and Najafi, Sh. 2015. Effect of nitric oxide on growth rate and some physiological indices of leaf seedlings planting in in vitro conditions, Journal of Cell and Texture, 6 (2): 203-195. (in Persian with English abstract)
21- Giannopolitis, C.N., and Reis, S.K. 1997. Superoxide dismutase I. Occurrence in higher plants. Plant Physiology, 59:309-314.
22- Haihua, H., Wenbiao, S., Maobing, Y., Langlai, X. 2002. Protective effects of nitric oxide on salt stress-induced oxidative damage to wheat (Triticum aestivum L.) leaves. Chinese Science Bulletin, 47: 677-681.
23- Jahromi, F., Aroca, R., Porcel, R., and Ruiz-Lozano, J.M. 2008. Influence of salinity on the in vitro development of Glomus intraradices and on the in vivo Physiological and molecular responses of mycorrhizal lettuce plants. Microbial Ecology, 55: 45-53.
24- Jeffries, P., and Barea, J.M. 2001. Arbuscular mycorrhiza-a key component of sustainable plant–soil ecosystems. In: Fungal associations (ed. Hock, B.) 95–113. Springer-Verlag, Berlin.
25- Jevremovic, S., Petric, M., Zivkovic, S., Trifunovic, M., and Subotic, A. 2010. Superoxide dismutase activity and isoenzyme profiles in bulbs of snake's head fritillary in response to cold treatment. Archives of Biological Sciences, 62: 553-558.
26- Kapoor, R., Giri, B., and Mukerji, K.G. 2004. Improved growth and essential oil yield and quality in (Foeniculum vulgare mill) on mycorrhizal inoculation supplemented with P-fertilizer. Bioresource Technology, 93: 307-311.
27- Khanahmadi, M.M, Naghdi Badi, H., Akhondzadeh, S., Khalighi-Sigaroodi, F., Mehrafarin, A., Shahriari, S. 2013. A Review of the medicinal plant of Glycyrrhiza glabra L. Journal of Medicinal Plants, 2(46):1-12.
28- Koroi, S.A.A. 1989. Gel elektrophers tische and spectral photometrischoe under uchungen zomein fiuss der temperature auf straktur and aktritat der amylase and peroxidase isoenzyme. Physiological Journal, 20: 15-23.
29- Kumar, S.G., Reddy, A.M., and Sudhakar, C. 2003. NaCl effects on proline metabolism in two high yielding genotypes of mulberry (Morus alba L.) with contrasting salt tolerance. Plant Science, 165:1245-1251.
30- Larose, G., Chenevert, R., Moutoglis, P., Gagne, S., Piche, Y., and Vierheilig, H. 2002. Flavonoid levels in roots of Medicago sativa L. are modulated by the developmental stage of the symbiosis and the root colonizing Arbuscular mycorrhizal fungus. Journal of Plant Physiology, 159:1329–1339.
31- Liu, X., Wang, L., Liu, L., Guo, Y., and Ren, H. 2011. Alleviating effect of exogenous nitric oxide in cucumber seedling against chilling stress. African Journal of Biotechnology, 10: 4380-4386.
32- Lu, S., Wang, Q., Li, G., Sun, S., Guo, Y., and Kuang, H. 2015. The treatment of rheumatoid arthritis using Chinese medicinal plants: from pharmacology to potential molecular mechanisms. Journal of Ethnopharmacology, 176:177-206.
33- Mafakheri, A., Siosemardeh, A., Bahramnejad, B., Struik, P.C., and Sohrabi, Y. 2011. Effect of drought stress and subsequent recovery on protein, carbohydrate contents, catalase and peroxidase activities in three chickpeas (Cicer arietinum) cultivars. Australian Journal of Crop Science, 10: 1255-1260.
34- Misra, N., and Saxena, P. 2009. Effect of salicylic acid on proline metabolism in lentil grown under salinity stress. Plant Science, 177: 181-188.
35- Nasibi, F., Yaghubi, M.M., and Manoochehri Kalantari. Kh. 2011. Comparison of the effect of sodium nitro process and arginine pre-treatment on some physiological responses of tomato (Lycopersicon esculentum) under water stress. Iran Biological Journal, 24 (6): 132-121.
36- Nayyar, H. 2003. Acclimation of osmolytes and osmotic adjustment in water-stressed wheat and maize as affected by calcium and its antagonists. Environmental and Experimental Botany, 50: 253-264.
37- Neill, S., Radhika, D., and Hancock, J. 2003. Nitric oxide signaling in the plant. New phytology, 159:11-35.
38- Nofal, O.A., and Rezk, A.L. 2009. Role of fertilization in improving quality of some agricultural crops. International Journal of Academic Research, 1: 59-65.
39- Nunez, M., Mazzafera, P., Mazorra, L.M., Siquira, W.J., and Zullo, M.A. 2003. Influence of a brassinosteroid analogue on antioxidant enzymes in rice grown in culture medium with NaCl. Plant Biology, 47: 67-70.
40- Ohkawa, H., Ohishi, N., and Yagi, K. 1979. Assay for lipid peroxidation in animal tissues by thiobarbituric acid reaction. Analytical Biochemistry, 95: 351.
41- Palavan, U.N., and Arisan, D. 2009. Nitric oxide signaling in plants. Biology Reviews, 75:203-229.
42- Panwar, J., and Tarafdar, J.C. 2006. Arbuscular mycorrhiza fungal dynamics under Mitragyna parvifolia (Roxb.) north. In Thar Desert. Applied soil Ecology, 34: 200-208.
43- Parrida, A.K., Das, A.B., and Mittra, B. 2004. Effects of salt on growth, ion accumulation photosynthesis and leaf anatomy of the mangrove, Bruguier parviflora. Trees Journal, 18:167-174.
44- Pereira, G.J.G., Molina, S.M.G., and Lea, P.J. 2002. The activity of antioxidant enzyme in response to cadmium in Crotalaria juncea. Plant Soil, 239: 123-132.
45- Rafiei, A.l., Husseini, M., Tadion, M., and Mazhari, M. 2014. The Effect of dormancy break treatments on seed germination of Licorice. Journal of Crop Improvements, 16 (4): 817-809. (in Persian with English abstract).
46- Rajaravindran, M., and Natarajan, S. 2012. Effects of salinity stress on growth and antioxidant enzymes of the halophyte Sesuvium portulacastrum. International Journal of Research in Plant Science, 2(1): 23-28.
47- Saleh, B. 2013. Water status and protein pattern change towards salt stress in Cotton. Journal of Stress Physiology & Biochemistry, 9(1): 113-123.
48- Sevengor, S., Yasar, F., Kusvuran, S., and Ellialtioglu, S. 2011. The effect of salt stress on growth, chlorophyll content, lipid peroxidation and antioxidative enzymes of pumpkin seedling. African Journal of Agricultural Research, 6(21):4920- 4924.
49- Shi, Q., Ding, F., Wang, X., and Wei, M. 2007. Exogenous nitric oxide protects cucumber roots against oxidative stress induced by salt stress. Plant Physiology and Biochemistry, 45: 542-550.
50- Singh, N.B., Kavita, Y., and Nimisha, A. 2014. Positive effects of nitric oxide on Solanum lycopersicum. Journal of Plant Interactions, 9: 10-18.
51- Stavros, D.V., Baodong, C., and Matthias, C.R. 2012. Arbuscular mycorrhiza and soil nitrogen cycling. Journal of Soil Biology and Biochemistry, 46: 53-62.
52- Swain, T., and Hillis, W.E. 1959. The phenolic constituents of Prunus domestica I. The quantitative analysis of phenolic constituents. Journal of Science and Food Agriculture, 10:63-68.
53- Tian, X., and Li, Y. 2006. Nitric oxide treatment alleviates drought stress in wheat seeding. Plant Biology, 50: 775-778.
54- Toussaint, J.P, Smith, F.A., and Smith, S.E. 2007. Arbuscular mycorrhizal fungi can induce the production of phytochemicals in sweet basil irrespective of phosphorus nutrition. Mycorrhiza, 17:291–297.
55- Wu, C.H., Tewari, R.K., Hahn, E.J., and Paek, K.Y. 2007. Nitric oxide elicitation induces the accumulation of secondary metabolites and antioxidant defense in adventitious roots of Echinacea purpura. Journal of Plant Biology, 50: 636–643.
56- Wu, X., Zhu, W., Zhang, H., Ding, H., and Zhang, H.J. 2011. Exogenous nitric oxide protects against salt-induced oxidative stress in the leaves from two genotypes of tomato (Lycopersicum esculentum Mill.). Acta Physiologiae Plantarum, 33:1199-1209.
57- Younesi, A., and Moradi, A.S. 2016. Evaluation of antioxidant enzymes activity in response to mycorrhizal inoculation in wheat under salt stress. Journal of Crop Improvement, 18 (1): 30-21. (in Persian with English abstract).
58- Yu, L., Gao, R., Shi, Q., Wang, X., Wei, M., and Yang, F. 2013. Exogenous application of sodium nitroprusside alleviated cadmium induced chlorosis, photosynthesis inhibition and oxidative stress in cucumber. Pakistan Journal of Botany, 45: 813-819
59- Zeng, C.L., Liu, L., Wang, B.R., Wu, X.M., and Zhou, Y. 2011. Physiological effects of exogenous nitric oxide on Brassica juncea seedlings under NaCl stress. Biologia Plantarum, 55: 345-348.
60- Zhang, Y.J., Zhang, X., Chen, C.J., Zhou, M.J., and Wang, H.C. 2010. Effects of fungicides JS399-19, azoxystrobin, tebuconazloe, and carbendazim on the physiological and biochemical indices and grain yield of winter wheat. Pesticide Biochemistry and Physiology, 98: 151–157.