The pharmaceutical properties of artemisia are partially attributed to their essential oils, containing a wide variety of active phytochemicals, such as monoterpenes, sesquiterpenes and triterpenes. Among important terpens are β-amyrin, β-caryophyllene, Linalool and β-Pinene that are used as anticancer, analgesic, antimicrobial, antioxidant and anti-inflammatory. In this study, we have examined the relative expression levels of four genes of β-amyrin synthase, β-caryophyllene synthase, Linalool Synthase, and β-Pinene Synthase in eight species of Artemisia by real-time PCR. We used 18S rRNA as a control gene.Maximum and minimum expression levels of β-amyrin synthase, β- caryophyllene synthase, Linalool Synthase and β-Pinene Synthase were observed related to A. annua at the flowering stage of A. scoparia (1.62) and the seedling stage of A. sieberi (-50.85), the flowering stage of A.campestris (56.28) and the flowering stage of A. vulgaris (-10.74), the budding stage of A. scoparia (15.86) and the budding stage of A. sieberi (-20), the budding stage of A.campestris (53.57) and the budding stage of A. diffusa (-23.49), respectively.

Zinc as an essential element for plant growth, plays an important role in many metabolic processes of plants, such as limonene synthase gene expression pathway. Limonene synthase is considered as an important enzyme in volatile oil biosynthesis that plays significant role in plant defensive reactions. In the present study, limonene synthase gene expression in Cumin was evaluated in presence of different concentration of Zinc elicitors based on a completely randomized design. Total RNA was extracted and cDNA synthesis was performed and Real Time PCR quantity assay method was used for limonene synthase gene expression. Results of the comparison of gene expression with Duncan's multiple range tests in different concentrations showed that in treated plants, there was a direct correlation between increasing of Zinc concentration up to 100 µM and limonene synthase gene expression. Limonene synthase gene expression by increasing production of monoterpens may produce more volatile oils in plant species and thus protects the plant against herbivores.

To investigate the effects of nano-chelate spraying of Iron, chemical -synthesis and green-synthesis silver nanoparticles on the expression of two genes, germacrene A synthase (TpGAS) and parthenolide synthase (TpPTS) genes which are involved in the parthenolide biosynthesis pathway of Tanacetum parthenium L., a factorial experiment based on randomized complete block design was conducted with three replications. The experiment took place in the greenhouse of Zabol University in Iran. At first, the total RNA from samples of leaves, and then the synthesis of cDNA were extracted. Afterward, Real Time PCR analysis was used to determine gene expression patterns. Parthenolide concentration was measured by high-performance liquid chromatography (HPLC). The analysis of the data was conducted by SAS software, and there was a significant increase in the expressions of both germacrene A synthase (TpGAS) and parthenolide synthase (TpPTS) genes of treated plants with green-synthesis silver nanoparticles in compare with the control plants, and some plants which were treated by chemical- synthesis silver nanoparticles and nano-chelate of Iron. Furthermore, the maximum expressions of both germacrene A synthase (TpGAS) and parthenolide synthase (TpPTS) genes were identified in groups of plants which were under the influence of drought stress, or were treated by green-synthesis silver nanoparticles. A positive relation was seen between the expressions of germacrene A synthase genes (TpGAS) and parthenolide synthase (TpPTS) genes with the amount of parthenolide itself. It can be concluded that green-synthesis silver nanoparticles and water deficit stress can cause an increase on the expressions of parthenolide and germacrene A synthase genes, and as result of that, cause an increase on parthenolide production of Tanacetum parthenium.

Black cumin (Nigella sativa) is an important medicinal plant belonging to the Ranunculaceae which produces large diversity of secondary metabolites especially terpenes. Mono (C10) and tri-terpenes (C30) are two major structural classes of terpene compounds. Several genes are involved in biosynthetic pathway of the monoterpenes known as MEP pathway، located in plastids. In the MEP pathway، monoterpene synthases are rate- limiting enzymes for monoterpene biosynthesis. In the biosynthetic pathway of monoterpenes geranyl diphosphate synthase catalyzing geranyl diphosphate (GDP) biosynthesis as universal precursor of monoterpenes. Oxidosqualenecyclases is of the most important. In this study gene expression patterns of a monoterpene synthase، GDP synthase، beta amirin synthase and squalene oxidase were evaluated under salicylic acid treatment. Gene expression analysis of the genes at trasnscript level using semi-quantitative PCR in response to salicylic acid treatment revealed their differential expression.

Dracocephalum kotschyi (Lamiaceae), as one of the remarkable aromatic plants, widely grows and also is cultivated in various temperate regions of Iran. There are diverse reports about the composition of the oil of this plant representing limonene derivatives as its major compounds. There is no report on cloning of mono- or sesquiterpene synthases from this plant. In the present study, the aroma profile of D. kotschyi has been extracted and analyzed via Headspace Solid-Phase Microextraction technique coupled with Gas Chromatography- Mass Spectroscopy. In order to determine the sequence of the active terpene synthase in this plant, first mRNA was prepared and cloning was performed by 3’ and 5’-RACEs-PCR method, then cDNA was sequenced and finally aligned with other recognized terpene synthases. The results showed that the plant leaves mainly comprised geranial (37.2%), limonene-10-al (28.5%), limonene (20.1%) and 1,1-dimethoxy decane (14.5%). Sequencing the cDNA cloned from this plant revealed the presence of a monoterpene synthase absolutely similar to limonene synthase, responsible in formation of limonene, terpinolene, camphene and some other cyclic monoterpenes in its young leaves.

Medicinal plants have had important roles in traditional medicine since ancient times. Secondary metabolites produced in plants have many applications in medicine, pharmacology and industry. The genus Artemisia is very important due to the production of sesquiterpene Artemisinin and many other compounds. Various sesquiterpenes production is catalyzed from central intermediate farnesyl diphosphate, by sesquiterpene enzymes. In this study seven Artemisia species local to Iran were planted. Flower tissues were sampled in flowering phase, in three biological replications. The relative expression of one prenyle transferase (Farnesyl diphophate synthase) and two sesquiterpene synthases (beta-caryophyllene synthase and beta-farnesene synthase) was studied by Real-time PCR. Artemisia annua was considered as the reference species. Besides, gas chromatography-mass spectrometry (GC-MS) was applied to investigate the end products produced by each terpene synthases. The results showed that the expression of terpene synthase genes is consistent with their products obtained in GC-MS. The maximum level of expression for terpene synthase-encoding genes was achieved for A. scoparia. Moreover, the results from GC-MS indicated the highest amount of production of beta-caryophyllene and beta-farnesene in this species. It can be concluded that the biosynthesis pathways of sesquiterpenes may be regulated by its specific sesquiterpene synthase.

Squalene synthase (E.C, 2.5.1.21) catalyzes the reductive dimerization of two molecules of farnesyl diphosphate to squalene, so that it is involved in the first step in cholesterol biosynthesis. Inhibition of squalene synthase is under consideration as an approach of decreasing cholesterol levels in the prevention of cardiovascular disease. Therefore squalene synthase is attractive object for the treatment of hypercholesterolemia, hypertriglyceridemia and coronary heart disease. Understanding the interaction of squalene synthase binding site with inhibitors is crucial for the development of pharmaceutical agents. At this aim, computer aided drug design is an applicable method that can study theses interactions and describe significant characteristics for squalene synthase binding site recognition. In the present work, we applied the molecular docking approach for 9 inhibitors of squalene synthase. Autodock Tools 4.2 was used in order to prepare the docking runs. After the docking runs, the final binding modes of the inhibitors were selected on the basis of the highest score or in other words best fit corresponding to the complex with the lowest free energy of binding. The docking results indicate that all inhibitors bind to squalene synthase active site. Our results clearly show that non polar interactions play a significant role in determining the binding free energy. The results also demonstrated that the inhibitor binding site is a hydrophobic pocket that completely is surrounded by the hydrophobic residue. Also, it was found that the inhibitor 6 (E5700) have the lowest binding free energy (-9.01 kcal/mol). The docking results will be useful for the structure-based drug design and the development of the pharmaceutical agents to treatment of coronary heart disease.

Common sage (Salvia officinalis) is one of the most important medicinal and aromatic plants and possesses anticancer, antioxidant and antimicrobial properties. One of the essential oil components of the sage plant is monoterpene, 1,8-cineole, that is responsible for some of these effects. The effect of foliar application of chitosan (control, 0.0, 0.25 and 0.50 g/L) under different levels of water deficit (FC = 60, 75 and 100%) were investigated on the expression level of cineole synthase gene in the roots and accumulation of 1,8-cineole content in the leaves of the sage plants. The experiment was conducted as factorial based on completely randomized design with three replications. The level of gene expression was determined by real time PCR in plant roots and the 1,8-cineole content was identified by gas chromatography/mass spectrometry in leaves. Results indicated that the expression of studied gene and 1,8-cineole content were strongly up-regulated with water deficit. Furthermore, foliar application of chitosan (specially in 0.5 g/L) effectively up-regulated the cineole synthase gene expression in roots and 1,8-cineole content in the shoots. Overall, water deficit stress was more effective than chitosan in the up regulation of cineole synthase gene and 1,8-cineole synthase content. The maximum level of cineole synthase gene expression and 1,8-cineole content were observed in mild drought stress and 0.5 g/L chitosan condition. Slight water deficit stress had no effect on gene expression but the final product was increased in shoots. For the first time, our results indicated that water deficit stress and chitosan could increase cineole synthase gene expression in the roots of common sage. Therefore, chitosan along with water deficit probably increase 1,8-cineole content in shoots, in part, through increasing the expression level of 1,8-cineole synthase gene in roots.

RNA-seq can facilitate and accelerate the identification of key genes involved in the synthesis of the plant secondary metabolites. The fruits of the medicinal plant Ajowan (Trachyspermum ammi) contain 2-9 % essential oil which the monoterpenes thymol, γ-terpenine and para-cymene, are the major constituents. In the present study transcriptome analysis of Ajowan via RNA-seq by Illumina high-throughput technique was done. 42260830 high-quality clean reads were assembled into 68051 unigenes with the average length of 859.4 and N50 of 1257 bp. In order to identify unigenes involved in terpenoid backbone biosynthetic pathway, the sequences were uploaded on the KAAS database. On the basis of results, 30 genes in the pathway were identified including all the genes in two terpenoid backbone pathway (MEP and MVA) from the beginning of the pathway to IPP production. Enzymes involved in MEP pathway that were present in our dataset included 1-deoxy-D-xylulose-5-phosphate synthase (DXS), 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR), 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase(ispD), 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase (ispE), 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (ispF), 4-hydroxy-3-methylbut-2-enyl diphosphate synthase (gcpE), 4-hydroxy-3-methylbut-2-en-1-yl diphosphate reductase (ispH), isopentenyl-diphosphate delta isomerase, isoprene synthase (ispS), and geranyl-diphosphate synthase (GPS). Likewise, MEV pathway genes included acetyl-CoA acetyltransferase, HMG-CoA synthase, HMG-CoA reductase (HMGCR), phosphomevalonate kinase and mevalonate diphosphate decarboxylase. Identification of genes involved in biosynthetic pathways of terpenoid backbone will aid in future studies of the genes characterization, over-expression and metabolite engineering.

Important monoterpenes such as camphor and fenchol are components of lavender (Lavandula angustifolia) essential oil. Monoterpene synthases are suitable target genes for metabolic engineering in Lavender. This study was performed to analyze the expression and induction of the two key genes (borneol synthase and fenchol synthase) involved in monoterpenes biosynthetic pathway in lavender after elicitation by methyl jasmonate and salicylic acid. First, plants in the pre-flowering and post-flowering stages were treated separately by methyl jasmonate and salicylic acid, each at a concentration of 1 mM. Then, plant leaves were harvested 0 (control), 24, 48 and 72 hours after treatments for gene expression analysis. The results showed that the expression of these genes was affected by salicylic acid and methyl jasmonate treatment. In general, the expression of both genes in the pre-flowering stage was higher than that of post-flowering. Moreover, the expression of borneol synthase was more affected by methyl jasmonate, so that its expression trend was completely increased up to 72 hours. Given the increased expression of borneol synthase and fenchol synthase in plants treated with salicylic acid, and methyl jasmonate it could be expected that the content of valuable monoterpenes such as camphor and fenchol increase in lavender.