Document Type : Research Paper - Plant Breeding

Authors

1 PhD Student in Production Engineering and Plant Genetics, Assistant Professor, Department of Plant Production and Genetics, Faculty of Agriculture, Shahid Chamran University of Ahvaz, Ahvaz, Iran

2 Assistant Professor, Department of Plant Production and Genetics, Faculty of Agriculture, Shahid Chamran University of Ahvaz, Ahvaz, Iran

3 Key Laboratory of Plant Resources/Beijing Botanical Garden, Institute of Botany, Chinese Academy of Sciences, Beijing, China

Abstract

Introduction
Iranian Kakoti (Ziziphora persica Bunge) which belongs to the mint genus is widely distributed in Iran. In some parts of Iran, it is used as a spice and condiment in food. Iranian Kakoti is also a medicinal plant with antioxidant and antimicrobial properties. Iranian Kakoti is used in traditional medicine as a stomach disinfectant and cold remedy.  Since the effect of stresses on the increase of secondary metabolites of plants has been proven, the aim of this study was to investigate the effect of drought stress on the secondary metabolites of Iranian Kakoti in comparison with normal conditions by assessment of the expression of genes which are related to the most important secondary metabolites of the studied plant. In previous studies, it was found that pulegone has the largest share among the metabolites produced in this plant.
 
 
 
Materials and Methods
Using the KEGG database, the metabolic pathway and genes involved in the production of the pulegone include the four key genes Isopiperitenol dehydrogenase, Limonene synthase, limonene hydroxylase and Isopiperitenone reductase were identified. Due to the lack of genomic information of Iranian Kakoti, the sequence of the four studied genes was predicted by the sequences of these genes in families close to this plant. The predicted sequences were used for primer design. The plants were cultivated under drought stress and normal conditions. The stress condition was achieved by reaching 30% Field Capacity. After observing phenotypic changes due to stress, the amount of proline and also Leaf relative water content (RWC) measured for the plants in both control and stress condition to confirm the accruing of the stress. Then leaf samples were collected. RNA extraction and cDNA synthesis were performed according to the manufacturer's instructions. The quantity and quality of the obtained RNA were evaluated by Nanodrop and gel electrophoresis. The qualified RNA samples were used for cDNA synthesis according to the manufacturer's protocols. Then the comparing the genes expression under drought stress with the control sample was performed.
 
Results and Discussion
In response to drought stress, leaf RWC was decreased to 31% in the plants under stress condition and proline levels increased by 1.9-fold compared to control value. Analysis of real-time PCR data showed that. The expressions of limonene synthase, limonene hydroxylase, Isopiperitenon dehydrogenase, Isopiperitenon reductase were increased 115, 400, 50 and 120 folds compared to control respectively.
 
Conclusion
The measurement of leaf RWC and proline were found to be beneficial methods to detection of incidence of drought stress in plant.  The expression of genes involved in pulegone synthesis compared to the control plants changes with drought stress. The implement of drought stress is a useful method to increase essential oil in Iranian Kakoti.
 

Keywords

Main Subjects

Abdalla, M., & El-Khoshiban, N. H. (2007). The influence of water stress on growth, relative water content, photosynthetic pigments, some metabolic and hormonal contents of two Triticium aestivum cultivars. Journal of Applied Sciences Research, 3(12), 2062-2074.
Abdollahi Mandoulakani, B., Alipoor, M., Darvishzadeh, R., & Majroomi Senji, B. (2017). The effect of drought stress on the expression of genes encoding phenylalanine ammonialyase (PAL), eugenol synthase 1 (EGS1) and caffeic acid O-methyltransferase (COMT) enzymes in Basil (Ocimum basilicum). Scientific Information Database, 9(4), 117-128. [In Persian]
Ahmadi Khoei, M., & Shabani, L. (2015). The effect of inoculation with mycorrhizal arbuscular fungion expression of limonene synthase in Mentha spicata L. genotypes. Iranian Journal of Plant Biology, 7(23), 51-62. [In Persian]
Baher, Z. F., Mirza, M., Ghorbanli, M., & Bagher Rezaii, M. (2002). The influence of water stress on plant height, herbal and essential oil yield and composition in Satureja hortensis L. Flavour and Fragrance Journal, 17(4), 275-277.
Chitsaz, M., Pargar, A., Naseri, M., Bazargan, M., & Ansari, M. (2007). Composition and anti- bacterial effects of hydro-alcoholic extract and essential oil of Ziziphora clinopodioides on selected bacteria. Scientific Journal of Shahed University, 68, 15-22.
Christou, A., Georgiadou, E. C., Filippou, P., Manganaris, G. A., & Fotopoulos, V. (2014). Establishment of a rapid, inexpensive protocol for extraction of high quality RNA from small amounts of strawberry plant tissues and other recalcitrant fruit crops. Gene, 537(1), 169-173.
Croteau, R. B., Davis, E. M., Ringer, K. L., & Wildung, M. R. (2005). Menthol biosynthesis and molecular genetics. Naturwissenschaften, 92(12), 562-577.
Duru, M. E., Ozturk, M., Ugur, A., & Ceylan, O. (2004). The constituents of essential oil and in vitro antimicrobial activity of Micromeria cilicica from Turkey.  Journal of Ethnopharmacology, 94(1), 43-48.
Fatima, S., Farooqi, A. A., & Sharma, S. (2002). Physiological and metabolic responses of different genotypes of Cymbopogon martinii and C. winterianus to water stress. Plant Growth Regulation, 37(2), 143-149.
Hassanpour, H., Khavari-Nejad, A., Niknam V, Razavi K., & Najafi F. (2014). Effect of penconazole and drought stress on the essential oil composition and gene expression of Mentha pulegium L. (Lamiaceae) at flowering stage. Acta Physiologiae Plantarum, 36(5), 1167-1175.
Hassanpour, H., Khavari-Nejad, R. A., Niknam, V., Najafi, F., & Razavi, K. (2012). Effects of penconazole and water deficit stress on physiological and antioxidative responses in pennyroyal (Mentha pulegium L.). Acta Physiologiae Plantarum, 34(4), 1537-1549.
Karp, F., Mihaliak A, Harris, L., & Croteau, R. (1990). Monoterpene biosynthesis specificity of the hydroxylations of -limonene by enzyme preparations from peppermint (Mentha piperita), spearmint (Mentha spicata), and perilla (Perilla frutescens) leaves. Archives of Biochemistry and Biophysics, 276(1), 219-226.
Khorasaninejad, S., Mousavi, A., Soltanloo, H., Hemmati, K., & Khalighi, A. (2011). The effect of drought stress on growth parameters, essential oil yield and constituent of Peppermint (Mentha piperita L.). Journal of Medicinal Plants Research, 5(22), 5360-5365.
Kjonaas, B., Venkatachalam, V., & Croteau, R. (1985). Metabolism of monoterpenes: Oxidation of isopiperitenol to isopiperitenone, and subsequent isomerization to piperitenone by soluble enzyme preparations from peppermint (Mentha piperita) leaves. Archives of Biochemistry and Biophysics, 238(1), 49-60.
Kjonaas, R., & Croteau, R. (1983). Demonstration that limonene is the first cyclic intermediate in the biosynthesis of oxygenated p-menthane monoterpenes in Mentha piperita and other Mentha species. Archives of Biochemistry and Biophysics, 220(1), 79-89.
Kleinwachter, M., & Selmar, D. (2015). New insights explain that drought stress enhances the quality of spice and medicinal plants: potential applications. Agronomy for Sustainable Development, 35(1), 121-131.
Lange, M., Wildung, R., Stauber, J., Sanchez, C., Pouchnik, D., & Croteau, R. (2000). Probing essential oil biosynthesis and secretion by functional evaluation of expressed sequence tags from mint glandular trichomes. Proceedings of the National Academy of Sciences of the United States of America, 97(6), 2934-2939.
Mirzaei, F., Ismaili, A., Fatehi, F., Ghaderi, A., & Afkar, S. (2019). The study on gene expression of 3-Hydroxy-3-Methylglutaryl-Coa Reductase (HMGR) and γ-terpinene synthesis in thymus vulgaris L. Under Water Deficit Stress.  Plant Productions, 42(2), 165-180. [In Persian]
Mozaffarian, V. (1996). A dictionary of Iranian plant names. Tehran: Farhang Moaser. [In Persian]
Negueruela, V. A., & Mata, R. M. (1986). The volatile oil of Ziziphora hispanica L. Flavour and Fragrance Journal, 1(3), 111-113.
Omidbaigi, R. (2008). Production and Processing of medicinal plants (Vol 3). Mashhad: Astan'e Qods'e Razavi publication. [In Persian]
Ozturk, S., & Ercisli, S. (2006). The chemical composition of Essential oil and in vitro antibacterial of essenential oils and methanol extract of Ziziphora persica bunge. Ehtanopharmacology, 106(3), 327-376.
Pirzad, A., Shakiba, M. R., Zehtab-Salmasi, S., Mohammadi, S. A., Darvishzadeh, R., & Samadi, A. (2011). Effect of water stress on leaf relative water content, chlorophyll, proline and soluble carbohydrates in Matricaria chamomilla L. Journal of Medicinal Plants Research, 5(12), 2483-2488.
Rahimi, Y., Taleei, A., & Ranjbar, M. (2017). Changes in the expression of key genes involved in the biosynthesis of menthol and menthofuran in Mentha piperita L. under drought stress. Acta Physiologiae Plantarum, 39(9), 1-9.
Rai, V., Vajpayee, P., Singh, S. N., & Mehrotra, S. (2004). Effect of chromium accumulation on photosynthetic pigments, oxidative stress defense system, nitrate reduction, proline level and eugenol content of Ocimum tenuiflorum L. Plant Science, 167(5), 1159-1169.
Rechinger, K. H., Browicz, K., Persson, K., & Wendelbo, P. (1982). Flora iranica, akademische druck-U. Verlagsanstalt, Graz, 150(2), 108-216.
Sadeghian, M., Hakimi, M. H., & Sodaeizadeh, H. (2015). Effect of drought stress on some physiological and morphological characteristics of Hymenocrater yazdianus. Journal of Biological and Environmental Sciences, 7, 110-119.
Soleymani, F., & Taheri, H. (2015). Effects of Gibberellic on gene expression of menthol biosynthesis pathway in Peppermint (Mentha piperita L.). International Journal of Advances in Chemical Engineering and Biological Sciences, 2(2), 131-134.
Zendehdel, M., & Babapour, V. (2010). Study of antinociceptive effevts of Ziziphora teniour and its interference on opioidergic and serotoninergic systems. Journal of Veterinary Research, 65(1), 57-60.