Expression Analysis of the Key Genes Involved in Biosynthesis Pathway of Major Component of Ziziphora persica Bunge Essential Oil under Drought Stress

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.

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.

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. 

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.